Selective delivery molecules and methods of use

ABSTRACT

Disclosed herein is a selective delivery molecule comprising: (a) an acidic sequence (portion A) which is effective to inhibit or prevent the uptake into cells or tissue retention, (b) a molecular transport or tissue retention sequence (portion B), and (c) a linker between portion A and portion B, and (d) cargo moieties (portion DA and DB).

CROSS REFERENCE

This application is a continuation of U.S. application Ser. No.16/264,296, filed Jan. 31, 2019, which is a continuation of U.S.application Ser. No. 15/685,942, filed on Aug. 24, 2017, now issued asU.S. Pat. No. 10,226,539 on Mar. 12, 2019, which is a continuation ofU.S. application Ser. No. 14/764,681, filed Jul. 30, 2015, now issued asU.S. Pat. No. 9,782,498 on Oct. 10, 2017, which is the National Stageentry of International Application No. PCT/US2014/013942, filed Jan. 30,2014, which claims the benefit of U.S. Provisional Application No.61/758,680, filed Jan. 30, 2013; each of which are incorporated hereinby reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on May 3, 2021, is named“AVS_008C3_SL.txt” and is 8,017 bytes in size.

SUMMARY OF THE INVENTION

Disclosed herein, in certain embodiments, is a selective deliverymolecule according to SDM-41. Disclosed herein, in certain embodiments,is a selective delivery molecule according to SDM-42. Disclosed herein,in certain embodiments, is a selective delivery molecule according toSDM-43. Disclosed herein, in certain embodiments, is a selectivedelivery molecule according to SDM-44. Disclosed herein, in certainembodiments, is a selective delivery molecule according to SDM-45.Disclosed herein, in certain embodiments, is a selective deliverymolecule according to SDM-46. Disclosed herein, in certain embodiments,is a selective delivery molecule according to SDM-47. Disclosed herein,in certain embodiments, is a selective delivery molecule according toSDM-48. Disclosed herein, in certain embodiments, is a selectivedelivery molecule according to SDM-49. Disclosed herein, in certainembodiments, is a selective delivery molecule according to SDM-50.Disclosed herein, in certain embodiments, is a selective deliverymolecule according to SDM-51. Disclosed herein, in certain embodiments,is a selective delivery molecule according to SDM-52. Disclosed herein,in certain embodiments, is a selective delivery molecule according toSDM-53. Disclosed herein, in certain embodiments, is a selectivedelivery molecule according to SDM-54. Disclosed herein, in certainembodiments, is a selective delivery molecule according to SDM-55.Disclosed herein, in certain embodiments, is a selective deliverymolecule according to SDM-56. Disclosed herein, in certain embodiments,is a selective delivery molecule according to SDM-57. Disclosed herein,in certain embodiments, is a selective delivery molecule according toSDM-58. Disclosed herein, in certain embodiments, is a selectivedelivery molecule according to SDM-59. Disclosed herein, in certainembodiments, is a selective delivery molecule according to SDM-60.Disclosed herein, in certain embodiments, is a selective deliverymolecule according to SDM-61. Disclosed herein, in certain embodiments,is a selective delivery molecule according to SDM-62. Disclosed herein,in certain embodiments, is a selective delivery molecule according toSDM-63. Disclosed herein, in certain embodiments, is a selectivedelivery molecule according to SDM-64. Disclosed herein, in certainembodiments, is a selective delivery molecule according to SDM-65.

Disclosed herein, in certain embodiments, are tissue samples comprisinga molecule selected from: SDM-41, SDM-42, SDM-43, SDM-44, SDM-45,SDM-46, SDM-47, SDM-48, SDM-49, SDM-50, SDM-51, SDM-52, SDM-53, SDM-54,SDM-55, SDM-56, SDM-57, SDM-58, SDM-59, SDM-60, SDM-61, SDM-62, SDM-63,SDM-64, or SDM-65. In some embodiments, the molecule is SDM-41. In someembodiments, the tissue sample is a pathology slide or section. In someembodiments, the tissue sample is cancerous. In some embodiments, thecancerous tissue is: breast cancer tissue, colorectal cancer tissue,squamous cell carcinoma tissue, skin cancer tissue, prostate cancertissue, melanoma tissue, thyroid cancer tissue, ovarian cancer tissue,or cancerous lymph node tissue. In some embodiments, the canceroustissue is breast cancer tissue. In some embodiments, the canceroustissue is colorectal cancer tissue. In some embodiments, the canceroustissue is cancerous lymph node tissue. In some embodiments, thecancerous tissue is squamous cell carcinoma tissue. In some embodiments,the cancerous tissue is skin cancer tissue.

Disclosed herein, in certain embodiments, are methods of delivering apair of imaging agents to a tissue of interest, comprising contactingthe tissue of interest with a molecule selected from: SDM-41, SDM-42,SDM-43, SDM-44, SDM-45, SDM-46, SDM-47, SDM-48, SDM-49, SDM-50, SDM-51,SDM-52, SDM-53, SDM-54, SDM-55, SDM-56, SDM-57, SDM-58, SDM-59, SDM-60,SDM-61, SDM-62, SDM-63, SDM-64, or SDM-65. In some embodiments, themolecule is SDM-41. In some embodiments, the tissue of interest iscancerous. In some embodiments, the cancerous tissue is: breast cancertissue, colorectal cancer tissue, squamous cell carcinoma tissue, skincancer tissue, prostate cancer tissue, melanoma tissue, thyroid cancertissue, ovarian cancer tissue or cancerous lymph node tissue. In someembodiments, the cancerous tissue is breast cancer tissue. In someembodiments, the cancerous tissue is colorectal cancer tissue. In someembodiments, the cancerous tissue is cancerous lymph node tissue. Insome embodiments, the cancerous tissue is squamous cell carcinomatissue. In some embodiments, the cancerous tissue is skin cancer tissue.

Disclosed herein, in certain embodiments, are methods of visualizing atissue of interest in an individual in need thereof, comprising: (a)administering to the individual a molecule selected from: SDM-41,SDM-42, SDM-43, SDM-44, SDM-45, SDM-46, SDM-47, SDM-48, SDM-49, SDM-50,SDM-51, SDM-52, SDM-53, SDM-54, SDM-55, SDM-56, SDM-57, SDM-58, SDM-59,SDM-60, SDM-61, SDM-62, SDM-63, SDM-64, or SDM-65; and (b) visualizingat least one of the imaging agents. In some embodiments, the molecule isSDM-41. In some embodiments, the tissue is cancerous. In someembodiments, the cancerous tissue is: breast cancer tissue, colorectalcancer tissue, squamous cell carcinoma tissue, skin cancer tissue,prostate cancer tissue, melanoma tissue, thyroid cancer tissue, ovariancancer tissue, or cancerous lymph node tissue. In some embodiments, thecancerous cell or tissue is breast cancer tissue. In some embodiments,the cancerous cell or tissue is colorectal cancer tissue. In someembodiments, the cancerous cell or tissue is cancerous lymph nodetissue. In some embodiments, the cancerous cell or tissue is squamouscell carcinoma tissue. In some embodiments, the cancerous cell or tissueis skin cancer tissue. In some embodiments, the method further comprisessurgically removing the tissue of interest from the individual. In someembodiments, the surgical margin surrounding the tissue of interest isdecreased. In some embodiments, the method further comprises preparing atissue sample from the removed cell or tissue of interest. In someembodiments, the method further comprises staging the cancerous tissue.In some embodiments, the method further comprises visualizingFörsters/fluorescence resonance energy transfer between the fluorescentmoiety and a fluorescence-quenching moiety of the molecule.

Disclosed herein, in certain embodiments, are tissue samples comprisinga molecule of Formula I:

[c _(M)-M]-[[D_(A)-c _(A)]-(A-X-B)-[c _(B)-D_(B)]]  Formula I

-   -   wherein,        -   X is a cleavable linker;        -   A is a peptide with a sequence comprising 5 to 9 acidic            amino acids;        -   B is a peptide with a sequence comprising 7 to 9 basic amino            acids;        -   c_(A), c_(B), and c_(M) are independently 0-1 amino acid;        -   M is a polyethylene glycol (PEG) polymer; and        -   D_(A) and D_(B) are each independently an imaging agent;    -   wherein [c_(M)-M] is bound at any position on or between A, X,        and B, [D_(A)-c_(A)] is bound to any amino acid on A or X, and        [c_(B)-D_(B)] is bound to any amino acid on B; and    -   wherein the molecule of Formula I is selected from: SDM-41,        SDM-42, SDM-43, SDM-44, SDM-45, SDM-46, SDM-47, SDM-48, SDM-49,        SDM-50, SDM-51, SDM-52, SDM-53, SDM-54, SDM-55, SDM-56, SDM-57,        SDM-58, SDM-59, SDM-60, SDM-61, SDM-62, SDM-63, SDM-64, or        SDM-65.        In some embodiments, the tissue sample is a pathology slide or        section. In some embodiments, the tissue sample is cancerous. In        some embodiments, the cancerous tissue is: breast cancer tissue,        colorectal cancer tissue, squamous cell carcinoma tissue, skin        cancer tissue, prostate cancer tissue, melanoma tissue, thyroid        cancer tissue, ovarian cancer tissue, or cancerous lymph node        tissue. In some embodiments, the cancerous tissue is breast        cancer tissue. In some embodiments, the cancerous tissue is        colorectal cancer tissue. In some embodiments, the cancerous        tissue is cancerous lymph node tissue. In some embodiments, the        cancerous tissue is squamous cell carcinoma tissue. In some        embodiments, the cancerous tissue is skin cancer tissue.

Disclosed herein, in certain embodiments, are methods of delivering apair of imaging agents to a tissue of interest, comprising contactingthe tissue of interest with a molecule of Formula I:

[c _(M)-M]-[[D_(A)-c _(A)]-(A-X-B)-[c _(B)-D_(B)]]  Formula I

-   -   wherein,        -   X is a cleavable linker;        -   A is a peptide with a sequence comprising 5 to 9 acidic            amino acids;        -   B is a peptide with a sequence comprising 7 to 9 basic amino            acids;        -   c_(A), c_(B), and c_(M) are independently 0-1 amino acid;        -   M is a polyethylene glycol (PEG) polymer; and        -   D_(A) and D_(B) are each independently an imaging agent;    -   wherein [c_(M)-M] is bound at any position on or between A, X,        and B, [D_(A)-c_(A)] is bound to any amino acid on A or X, and        [c_(B)-D_(B)] is bound to any amino acid on B; and    -   wherein the molecule of Formula I is selected from: SDM-41,        SDM-42, SDM-43, SDM-44, SDM-45, SDM-46, SDM-47, SDM-48, SDM-49,        SDM-50, SDM-51, SDM-52, SDM-53, SDM-54, SDM-55, SDM-56, SDM-57,        SDM-58, SDM-59, SDM-60, SDM-61, SDM-62, SDM-63, SDM-64, or        SDM-65.        In some embodiments, the tissue of interest is cancerous. In        some embodiments, the cancerous tissue is: breast cancer tissue,        colorectal cancer tissue, squamous cell carcinoma tissue, skin        cancer tissue, prostate cancer tissue, melanoma tissue, thyroid        cancer tissue, ovarian cancer tissue, or cancerous lymph node        tissue. In some embodiments, the cancerous tissue is breast        cancer tissue. In some embodiments, the cancerous tissue is        colorectal cancer tissue. In some embodiments, the cancerous        tissue is cancerous lymph node tissue. In some embodiments, the        cancerous tissue is squamous cell carcinoma tissue. In some        embodiments, the cancerous tissue is skin cancer tissue.

Disclosed herein, in certain embodiments, are methods of visualizing atissue of interest in an individual in need thereof, comprising: (a)administering to the individual a molecule of Formula I that localizesto the tissue of interest in the individual,

[c _(M)-M]-[[D_(A)-c _(A)]-(A-X-B)-[c _(B)-D_(B)]]  Formula I

-   -   wherein,        -   X is a cleavable linker;        -   A is a peptide with a sequence comprising 5 to 9 acidic            amino acids;        -   B is a peptide with a sequence comprising 7 to 9 basic amino            acids;        -   c_(A), c_(B), and c_(M) are independently 0-1 amino acid;        -   M is a polyethylene glycol (PEG) polymer; and        -   D_(A) and D_(B) are each independently an imaging agent; and    -   wherein [c_(M)-M] is bound at any position on or between A, X,        and B, [D_(A)-c_(A)] is bound to any amino acid on A or X, and        [c_(B)-D_(B)] is bound to any amino acid on B;    -   wherein the molecule of Formula I is selected from: SDM-41,        SDM-42, SDM-43, SDM-44, SDM-45, SDM-46, SDM-47, SDM-48, SDM-49,        SDM-50, SDM-51, SDM-52, SDM-53, SDM-54, SDM-55, SDM-56, SDM-57,        SDM-58, SDM-59, SDM-60, SDM-61, SDM-62, SDM-63, SDM-64, or        SDM-65; and        -   (b) visualizing at least one of the imaging agents;            In some embodiments, the tissue of interest is cancerous. In            some embodiments, the cancerous tissue is: breast cancer            tissue, colorectal cancer tissue, squamous cell carcinoma            tissue, skin cancer tissue, prostate cancer tissue, melanoma            tissue, thyroid cancer tissue, ovarian cancer tissue, or            cancerous lymph node tissue. In some embodiments, the            cancerous tissue is breast cancer tissue. In some            embodiments, the cancerous tissue is colorectal cancer            tissue. In some embodiments, the cancerous tissue is            cancerous lymph node tissue. In some embodiments, the            cancerous tissue is squamous cell carcinoma tissue. In some            embodiments, the cancerous tissue is skin cancer tissue. In            some embodiments, the methods further comprise surgically            removing the tissue of interest from the individual. In some            embodiments, the surgical margin surrounding the tissue of            interest is decreased. In some embodiments, the methods            further comprise preparing a tissue sample from the removed            tissue of interest. In some embodiments, the methods further            comprise staging the cancerous tissue.

Disclosed herein, in certain embodiments, is a peptide according toPeptide P-16.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows that the velocity of MMP-7 cleavage of SDM-41 increaseswith increasing SDM-41 concentration, consistent with Michaelis-Mentenkinetics (Example 2a).

FIG. 2 depicts donor (left), acceptor (middle), and fluorescenceemission ratio (right) images for SDM-41 (Example 6a).

FIG. 3 shows a scatter plot of emission ratio data of positive andnegative nodes using SDM-41 in mouse metastatic lymph node model(Example 6b).

FIG. 4 shows a ROC curve generated by changing the threshold value usedto assign either a positive or negative metastatic prediction fromemission ratio data using SDM-41 in metastatic lymph node model (Example6b).

FIG. 5 depicts change in SDM-41 fluorescence ratio in homogenizedcancerous tissue (M1120909A2, M1121603A2, M1121797A6) and healthy tissue(M1120909B2, M1121797B6, M1121603B2) from breast cancer patients,individual kinetic traces (Example 8). The cancerous tissue (M112090A2,M1121603A2, and M1121797A6) cleaves SDM-41 faster than normal tissue((M112090B2, M1121603B2, and M1121797B6).

FIG. 6 exemplifies homogenized cancerous human breast tissue cleavingSDM-41 to a greater extent (˜3-fold) than adjacent healthy tissue fromthe same patient (Example 8).

DETAILED DESCRIPTION OF THE INVENTION

Selective delivery molecules (SDMs) allow the targeted delivery oftherapeutic agents and/or imaging agents to specific cells and/ortissues. In some embodiments, selective delivery molecules comprise (a)a molecular transport or tissue retention sequence (portion B), (b)cargo moieties (portion D_(A) and D_(B)) bound to portion A, B, or X,(c) X a linker, and (d) a macromolecular carrier and (e) an acidicsequence (portion A) which is effective to inhibit or prevent the uptakeinto cells or tissue retention. In some embodiments, cleavage of Xlinker, which allows the separation of portion A from portion B, iseffective to allow the uptake or retention of portion B and the attachedcargo into cells and tissue. However, selective delivery molecules maybe subject to rapid pharmacokinetic clearance with short plasmahalf-life, broad distribution, and slow wash out from multiplenon-target tissues with non-specific uptake. Thus, there is a need for aselective delivery molecule with increased in vivo circulation,accumulation in target tissue relative to non-target tissue, modulatedextravasation selectivity, and modulated bio-distribution. For imagingagents, there is a need for increased contrast in target tissue relativeto background tissue.

Certain Definitions

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

As used herein, the term “targeting molecule” refers to any agent (e.g.,peptide, protein, nucleic acid polymer, aptamer, or small molecule) thatassociates with (e.g., binds to) a target of interest. The target ofinterest may be a tissue, a cell, a cellular structure (e.g., anorganelle), a protein, a peptide, a polysaccharide, or a nucleic acidpolymer. In some embodiments, the targeting molecule is any agent thatassociates with (e.g., binds to) one or more cancer cells of a subject.

The term PEG means polyethylene glycol polymer. In some embodiments, thePEG is a polydisperse. In some embodiments, the PEG is discreet.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to naturally occurring amino acid polymers as well as aminoacid polymers in which one or more amino acid residues is anon-naturally occurring amino acid (e.g., an amino acid analog). Theterms encompass amino acid chains of any length, including full lengthproteins (i.e., antigens), wherein the amino acid residues are linked bycovalent peptide bonds. As used herein, the terms “peptide” refers to apolymer of amino acid residues typically ranging in length from 2 toabout 50 residues. In certain embodiments the peptide ranges in lengthfrom about 2, 3, 4, 5, 7, 9, 10, or 11 residues to about 50, 45, 40, 45,30, 25, 20, or 15 residues. In certain embodiments the peptide ranges inlength from about 8, 9, 10, 11, or 12 residues to about 15, 20 or 25residues. Where an amino acid sequence is provided herein, L-, D-, orbeta amino acid versions of the sequence are also contemplated as wellas retro, inversion, and retro-inversion isoforms. Peptides also includeamino acid polymers in which one or more amino acid residues is anartificial chemical analogue of a corresponding naturally occurringamino acid, as well as to naturally occurring amino acid polymers. Inaddition, the term applies to amino acids joined by a peptide linkage orby other modified linkages (e.g., where the peptide bond is replaced byan α-ester, a β-ester, a thioamide, phosphonamide, carbamate,hydroxylate, and the like (see, e.g., Spatola, (1983) Chem. Biochem.Amino Acids and Proteins 7: 267-357), where the amide is replaced with asaturated amine (see, e.g., Skiles et al., U.S. Pat. No. 4,496,542,which is incorporated herein by reference, and Kaltenbronn et al.,(1990) Pp. 969-970 in Proc. 11th American Peptide Symposium, ESCOMScience Publishers, The Netherlands, and the like)).

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide. Such analogs have modifiedR groups (e.g., norleucine) or modified peptide backbones, but retainthe same basic chemical structure as a naturally occurring amino acid.Amino acid mimetics refers to chemical compounds that have a structurethat is different from the general chemical structure of an amino acid,but that functions in a manner similar to a naturally occurring aminoacid. Amino acids are either D amino acids of L amino acids.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

One of skill will recognize that individual substitutions, deletions oradditions to a peptide, polypeptide, or protein sequence which alters,adds or deletes a single amino acid or a small percentage of amino acidsin the encoded sequence is a “conservatively modified variant” where thealteration results in the substitution of an amino acid with achemically similar amino acid. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and alleles of theinvention.

As used herein, the term “label” refers to a molecule that facilitatesthe visualization and/or detection of a targeting molecule disclosedherein. In some embodiments, the label is a fluorescent moiety.

The phrase “specifically binds” when referring to the interactionbetween a targeting molecule disclosed herein and a target (e.g.,purified protein, cancer cells or cancerous tissue, tumor, or metastaticlesion, metastases, or lymph node or metastatic lymph node), refers tothe formation of a high affinity bond between the targeting molecule andthe target. Further, the term means that the targeting molecule has lowaffinity for non-targets.

“Selective binding,” “selectivity,” and the like refers to thepreference of an agent to interact with one molecule as compared toanother. Preferably, interactions between a targeting molecule disclosedherein and a target are both specific and selective. Note that in someembodiments an agent is designed to “specifically bind” and “selectivelybind” two distinct, yet similar targets without binding to otherundesirable targets

The terms “individual,” “patient,” or “subject” are usedinterchangeably. As used herein, they mean any mammal (i.e. species ofany orders, families, and genus within the taxonomic classificationanimalia: chordata: vertebrata: mammalia). In some embodiments, themammal is a human. None of the terms require or are limited to situationcharacterized by the supervision (e.g. constant or intermittent) of ahealth care worker (e.g. a doctor, a registered nurse, a nursepractitioner, a physician's assistant, an orderly, or a hospice worker).

The terms “administer,” “administering”, “administration,” and the like,as used herein, refer to the methods that may be used to enable deliveryof agents or compositions to the desired site of biological action.These methods include, but are not limited to parenteral injection(e.g., intravenous, subcutaneous, intraperitoneal, intramuscular,intravascular, intrathecal, intravitreal, infusion, or local).Administration techniques that are optionally employed with the agentsand methods described herein, include e.g., as discussed in Goodman andGilman, The Pharmacological Basis of Therapeutics, current ed.;Pergamon; and Remington's, Pharmaceutical Sciences (current edition),Mack Publishing Co., Easton, Pa.

The term “pharmaceutically acceptable” as used herein, refers to amaterial that does not abrogate the biological activity or properties ofthe agents described herein, and is relatively nontoxic (i.e., thetoxicity of the material significantly outweighs the benefit of thematerial). In some instances, a pharmaceutically acceptable material maybe administered to an individual without causing significant undesirablebiological effects or significantly interacting in a deleterious mannerwith any of the components of the composition in which it is contained.

The term “surgery” as used herein, refers to any method that may be usedto investigate, manipulate, change, or cause an effect in a tissue by aphysical intervention. These methods include, but are not limited toopen surgery, endoscopic surgery, laparoscopic surgery, minimallyinvasive surgery, robotic surgery, and any procedures that may affect acancerous tissue such as tumor resection, cancer tissue ablation, cancerstaging, cancer diagnosis, lymph node staging, sentinel lymph nodedetection, or cancer treatment.

The term “guided surgery” as used herein, refers to any surgicalprocedure where the surgeon employs an imaging agent to guide thesurgery.

The term “cancer” as used herein, refers to any disease involvinguncontrolled growth or proliferation of cells in the human body. Cancersmay further be characterized by the ability of cells to migrate from theoriginal site and spread to distant sites (i.e., metastasize). Cancersmay be sarcomas, carcinomas, lymphomas, leukemias, blastomas, or germcell tumors. Cancers may occur in a variety of tissues including but notlimited to lung, breast, ovaries, colon, esophagus, rectum, bone,prostate, brain, pancreas, bladder, kidney, liver, blood cells, lymphnodes, thyroid, skin, and stomach.

Selective Delivery Molecules

Disclosed herein, in certain embodiments, is a selective deliverymolecule according to SDM-41. Disclosed herein, in certain embodiments,is a selective delivery molecule according to SDM-42. Disclosed herein,in certain embodiments, is a selective delivery molecule according toSDM-43. Disclosed herein, in certain embodiments, is a selectivedelivery molecule according to SDM-44. Disclosed herein, in certainembodiments, is a selective delivery molecule according to SDM-45.Disclosed herein, in certain embodiments, is a selective deliverymolecule according to SDM-46. Disclosed herein, in certain embodiments,is a selective delivery molecule according to SDM-47. Disclosed herein,in certain embodiments, is a selective delivery molecule according toSDM-48. Disclosed herein, in certain embodiments, is a selectivedelivery molecule according to SDM-49. Disclosed herein, in certainembodiments, is a selective delivery molecule according to SDM-50.Disclosed herein, in certain embodiments, is a selective deliverymolecule according to SDM-51. Disclosed herein, in certain embodiments,is a selective delivery molecule according to SDM-52. Disclosed herein,in certain embodiments, is a selective delivery molecule according toSDM-53. Disclosed herein, in certain embodiments, is a selectivedelivery molecule according to SDM-54. Disclosed herein, in certainembodiments, is a selective delivery molecule according to SDM-55.Disclosed herein, in certain embodiments, is a selective deliverymolecule according to SDM-56. Disclosed herein, in certain embodiments,is a selective delivery molecule according to SDM-57. Disclosed herein,in certain embodiments, is a selective delivery molecule according toSDM-58. Disclosed herein, in certain embodiments, is a selectivedelivery molecule according to SDM-59. Disclosed herein, in certainembodiments, is a selective delivery molecule according to SDM-60.Disclosed herein, in certain embodiments, is a selective deliverymolecule according to SDM-61. Disclosed herein, in certain embodiments,is a selective delivery molecule according to SDM-62. Disclosed herein,in certain embodiments, is a selective delivery molecule according toSDM-63. Disclosed herein, in certain embodiments, is a selectivedelivery molecule according to SDM-64. Disclosed herein, in certainembodiments, is a selective delivery molecule according to SDM-65.

Disclosed herein, in certain embodiments, are selective deliverymolecule of Formula I, having the structure:

[c _(M)-M]-[[D_(A)-c _(A)]-(A-X-B)-[c _(B)-D_(B)]]  Formula I

wherein,

-   -   X is a cleavable linker;    -   A is a peptide with a sequence comprising 5 to 9 acidic amino        acids;    -   B is a peptide with a sequence comprising 7 to 9 basic amino        acids;    -   c_(A), c_(B), and c_(M) are independently 0-1 amino acid;    -   M is a macromolecule carrier; and    -   D_(A) and D_(B) are each independently selected from an imaging        agent and a therapeutic; and        wherein [c_(M)-M] is bound at any position on or between A, X,        and B, [D_(A)-c_(A)] is bound to any amino acid on A or X, and        [c_(B)-D_(B)] is bound to any amino acid on B. In some        embodiments, A and B do not have an equal number of acidic and        basic amino acids. In some embodiments, the number of basic        amino acids in B is greater than the number of acidic amino        acids in A. In some embodiments, A is a peptide comprising 5 or        9 consecutive glutamates (SEQ ID NOS: 4-5, respectively. In some        embodiments, B is a peptide comprising 8 or 9 consecutive        arginines (SEQ ID NOS: 6-7, respectively). In some embodiments,        A is a peptide comprising 5 or 9 consecutive glutamates (SEQ ID        NOS: 4-5, respectively) and B is a peptide comprising 8 or 9        consecutive arginines (SEQ ID NOS: 6-7, respectively). In some        embodiments, A is a peptide comprising 5 consecutive glutamates        (SEQ ID NO: 4) and B is a peptide comprising 8 consecutive        arginines (SEQ ID NO: 6). In some embodiments, c_(A), c_(B), and        c_(M) are each independently a 0-1 amino acid. In some        embodiments, c_(A), c_(B), and c_(M) are each independently        selected from a naturally-occurring amino acid or a        non-naturally-occurring amino acid. In some embodiments, c_(A),        c_(B), and c_(M) are each independently selected from a D amino        acid, a L amino acid, an α-amino acid, a β-amino acid, or a        δ-amino acid. In some embodiments, c_(A), c_(B), and c_(M) are        each independently selected from any amino acid having a free        thiol group, any amino acid having a free amino group (e.g., a        N-terminal amine group), and any amino acid with a side chain        capable of forming an oxime or hydrazone bond upon reaction with        a hydroxylamine or hydrazine group. In some embodiments, c_(A),        c_(B), and c_(M) are each independently selected from        D-cysteine, D-glutamate, lysine, and para-4-acetyl        L-phenylalanine. In some embodiments, c_(B) is any amino acid        having a free thiol group. In some embodiments, c_(B) is        D-cysteine. In some embodiments, c_(A) is any amino acid having        a N-terminal amine group. In some embodiments, c_(A) is        D-glutamate. In some embodiments, c_(A) is lysine. In some        embodiments, c_(M) is any amino acid with a side chain capable        of forming an oxime or hydrazone bond upon reaction with a        hydroxylamine or hydrazine group. In some embodiments, c_(M) is        para-4-acetyl L-phenylalanine. In some embodiments, X is        cleavable by a protease. In some embodiments, X is cleavable by        a matrix metalloproteinase. In some embodiments, X comprises an        amino acid sequence that is cleavable by MMP2, MMP7, MMP9, or        MMP14. In some embodiments, X comprises a peptide linkage. In        some embodiments, X comprises an amino acid sequence selected        from: PLGLAG (SEQ ID NO: 2), PLG-C(me)-AG (SEQ ID NO:1),        RPLALWRS (SEQ ID NO: 3), ESPAYYTA (SEQ ID NO: 8), DPRSFL (SEQ ID        NO: 9), PPRSFL (SEQ ID NO: 10), RLQLKL (SEQ ID NO: 11), and        RLQLK(Ac) (SEQ ID NO: 12. In some embodiments, X comprises the        amino acid sequence PLGLAG (SEQ ID NO: 2). In some embodiments,        X comprises the amino acid sequence PLG-C(me)-AG (SEQ ID NO: 1).        In some embodiments, X comprises the amino acid sequence        RPLALWRS (SEQ ID NO: 3). In some embodiments, X comprises the        amino acid sequence DPRSFL (SEQ ID NO: 9). In some embodiments,        X comprises the amino acid sequence RLQLKL (SEQ ID NO:11). In        some embodiments, X comprises the amino acid sequence RLQLK(Ac)        (SEQ ID NO: 12). In some embodiments, M is selected from a        protein, a natural polymer, a synthetic polymer, or a dendrimer.        In some embodiments, M is selected from dextran, PEG polymers,        albumin, or a combination thereof. In some embodiments, M is PEG        polymers. In some embodiments, M is PEG polymers having an        average molecular weight of approximately 0.5 KDa (PEG 0.5 KDa),        2 kDa (PEG 2 KDa), 5 kDa (PEG 5 KDa), 12 kDa (PEG 12 kDa), 20        kDa (PEG 20 kDa), 30 kDa (PEG 30 kDa), and 40 kDa (PEG 40 kDa).        In some embodiments, D_(A) and D_(B) are a pair of donor and        acceptor fluorescent moieties that are capable of undergoing        Försters/fluorescence resonance energy transfer with the other.        In some embodiments, D_(A) and D_(B) are Cy5 and Cy7. In some        embodiments, D_(A) and D_(B) are Cy5 and IRDye750. In some        embodiments, D_(A) and D_(B) are Cy5 and IRDye800. In some        embodiments, D_(A) and D_(B) are Cy5 and ICG. In some        embodiments, D_(A) and D_(B) are a fluorescent moiety and a        fluorescence-quenching moiety. In some embodiments, the molecule        of Formula I is: SDM-41, SDM-42, SDM-43, SDM-44, SDM-45, SDM-46,        SDM-47, SDM-48, SDM-49, SDM-50, SDM-51, SDM-52, SDM-53, SDM-54,        SDM-55, SDM-56, SDM-57, SDM-58, SDM-59, SDM-60, SDM-61, SDM-62,        SDM-63, SDM-64, or SDM-65. In some embodiments, the molecule of        Formula I is SDM-41.

Disclosed herein, in certain embodiments, are selective deliverymolecules of Formula I, having the structure:

[c _(M)-M]-[[D_(A)-c _(A)]-(A-X-B)-[c _(B)-D_(B)]]  Formula I

wherein,

-   -   X is a cleavable linker;    -   A is a peptide with a sequence comprising 5 to 9 acidic amino        acids;    -   B is a peptide with a sequence comprising 7 to 9 basic amino        acids;    -   c_(A), c_(B), and c_(M) are independently 0-1 amino acid;    -   M is a polyethylene glycol (PEG) polymer; and    -   D_(A) and D_(B) are each independently an imaging agent; and    -   wherein [c_(M)-M] is bound to at any position on A or X,        [D_(A)-c_(A)] is bound to any amino acid on A or X, and        [c_(B)-D_(B)] is bound to any amino acid on B.        In some embodiments, A and B do not have an equal number of        acidic and basic amino acids. In some embodiments, the number of        basic amino acids in B is greater than the number of acidic        amino acids in A. In some embodiments, A is a peptide comprising        5 or 9 consecutive glutamates (SEQ ID NOS: 4-5, respectively).        In some embodiments, B is a peptide comprising 8 or 9        consecutive arginines (SEQ ID NOS: 6-7, respectively). In some        embodiments, A is a peptide comprising 5 or 9 consecutive        glutamates (SEQ ID NOS: 4-5, respectively) and B is a peptide        comprising 8 or 9 consecutive arginines (SEQ ID NOS: 6-7,        respectively). In some embodiments, A is a peptide comprising 5        consecutive glutamates (SEQ ID NO: 4) and B is a peptide        comprising 8 consecutive arginines (SEQ ID NO: 6). In some        embodiments, c_(A), c_(B), and c_(M) are each independently a        0-1 amino acid. In some embodiments, c_(A), c_(B), and c_(M) are        each independently selected from a naturally-occurring amino        acid or a non-naturally-occurring amino acid. In some        embodiments, c_(A), c_(B), and c_(M) are each independently        selected from a D amino acid, a L amino acid, an α-amino acid, a        β-amino acid, or a δ-amino acid. In some embodiments, c_(A),        c_(B), and c_(M) are each independently selected from any amino        acid having a free thiol group, any amino acid having a        N-terminal amine group, and any amino acid with a side chain        capable of forming an oxime or hydrazone bond upon reaction with        a hydroxylamine or hydrazine group. In some embodiments, c_(A),        c_(B), and c_(M) are each independently selected from        D-cysteine, D-glutamate, lysine, and para-4-acetyl        L-phenylalanine. In some embodiments, c_(B) is any amino acid        having a free thiol group. In some embodiments, c_(B) is        D-cysteine. In some embodiments, c_(A) is any amino acid having        a N-terminal amine group. In some embodiments, c_(A) is        D-glutamate. In some embodiments, c_(A) is lysine. In some        embodiments, c_(M) is any amino acid with a side chain capable        of forming an oxime or hydrazone bond upon reaction with a        hydroxylamine or hydrazine group. In some embodiments, c_(M) is        para-4-acetyl L-phenylalanine. In some embodiments, X is        cleavable by a protease. In some embodiments, X is cleavable by        a matrix metalloproteinase. In some embodiments, X comprises an        amino acid sequence that is cleavable by MMP2, MMP7, MMP9, or        MMP14. In some embodiments, X comprises a peptide linkage. In        some embodiments, X comprises an amino acid sequence selected        from: PLGLAG (SEQ ID NO: 2), PLG-C(me)-AG (SEQ ID NO: 1),        RPLALWRS (SEQ ID NO: 3), ESPAYYTA (SEQ ID NO: 8), DPRSFL (SEQ ID        NO:9), PPRSFL (SEQ ID NO: 10), RLQLKL (SEQ ID NO: 11), and        RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, X comprises the        amino acid sequence PLGLAG (SEQ ID NO: 2). In some embodiments,        X comprises the amino acid sequence PLG-C(me)-AG (SEQ ID NO: 1).        In some embodiments, X comprises the amino acid sequence        RPLALWRS (SEQ ID NO: 3). In some embodiments, X comprises the        amino acid sequence DPRSFL (SEQ ID NO: 9). In some embodiments,        X comprises the amino acid sequence PPRSFL (SEQ ID NO: 10). In        some embodiments, X comprises the amino acid sequence RLQLKL        (SEQ ID NO: 11). In some embodiments, X comprises the amino acid        sequence RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, D_(A)        and D_(B) are a pair of acceptor and donor fluorescent moieties        that are capable of undergoing Försters/fluorescence resonance        energy transfer with the other. In some embodiments, D_(A) and        D_(B) are Cy5 and Cy7. In some embodiments, D_(A) and D_(B) are        Cy5 and IRDye750. In some embodiments, D_(A) and D_(B) are Cy5        and IRDye800. In some embodiments, D_(A) and D_(B) are Cy5 and        ICG. In some embodiments, D_(A) and D_(B) are a fluorescent        moiety and a fluorescence-quenching moiety. In some embodiments,        the molecule of Formula I is: SDM-41, SDM-42, SDM-43, SDM-44,        SDM-45, SDM-46, SDM-47, SDM-48, SDM-49, SDM-50, SDM-51, SDM-52,        SDM-53, SDM-54, SDM-55, SDM-56, SDM-57, SDM-58, SDM-59, SDM-60,        SDM-61, SDM-62, SDM-63, SDM-64, or SDM-65. In some embodiments,        the molecule of Formula I is SDM-41.

Portion A

In some embodiments, A is a peptide with a sequence comprising 2 to 20acidic amino acids. In some embodiments, peptide portion A comprisesbetween about 2 to about 20 acidic amino acids. In some embodiments,peptide portion A comprises between about 5 to about 20 acidic aminoacids. In some embodiments, A has a sequence comprising 5 to 9 acidicamino acids. In some embodiments, A has a sequence comprising 5 to 8acidic amino acids. In some embodiments, A has a sequence comprising 5to 7 acidic amino acids. In some embodiments, A has a sequencecomprising 5 acidic amino acids. In some embodiments, A has a sequencecomprising 6 acidic amino acids. In some embodiments, A has a sequencecomprising 7 acidic amino acids. In some embodiments, A has a sequencecomprising 8 acidic amino acids. In some embodiments, A has a sequencecomprising 9 acidic amino acids.

In some embodiments, peptide portion A comprises between about 2 toabout 20 consecutive acidic amino acids. In some embodiments, peptideportion A comprises between about 5 to about 20 consecutive acidic aminoacids. In some embodiments, A has a sequence comprising 5 to 9consecutive acidic amino acids. In some embodiments, A has a sequencecomprising 5 to 8 consecutive acidic amino acids. In some embodiments, Ahas a sequence comprising 5 to 7 consecutive acidic amino acids. In someembodiments, A has a sequence comprising 5 consecutive acidic aminoacids. In some embodiments, A has a sequence comprising 6 consecutiveacidic amino acids. In some embodiments, A has a sequence comprising 7consecutive acidic amino acids. In some embodiments, A has a sequencecomprising 8 consecutive acidic amino acids. In some embodiments, A hasa sequence comprising 9 consecutive acidic amino acids.

In some embodiments, peptide portion A comprises between about 2 toabout 20 acidic amino acids selected from, aspartates and glutamates. Insome embodiments, peptide portion A comprises between about 5 to about20 acidic amino acids selected from, aspartates and glutamates. In someembodiments, A has a sequence comprising 5 to 9 acidic amino acidsselected from, aspartates and glutamates. In some embodiments, A has asequence comprising 5 to 8 acidic amino acids selected from, aspartatesand glutamates. In some embodiments, A has a sequence comprising 5 to 7acidic amino acids selected from, aspartates and glutamates. In someembodiments, A has a sequence comprising 5 acidic amino acids selectedfrom, aspartates and glutamates. In some embodiments, A has a sequencecomprising 6 acidic amino acids selected from, aspartates andglutamates. In some embodiments, A has a sequence comprising 7 acidicamino acids selected from, aspartates and glutamates. In someembodiments, A has a sequence comprising 8 acidic amino acids selectedfrom, aspartates and glutamates. In some embodiments, A has a sequencecomprising 9 acidic amino acids selected from, aspartates andglutamates.

In some embodiments, peptide portion A comprises between about 2 toabout 20 consecutive acidic amino acids selected from, aspartates andglutamates. In some embodiments, peptide portion A comprises betweenabout 5 to about 20 consecutive acidic amino acids selected from,aspartates and glutamates. In some embodiments, A has a sequencecomprising 5 to 9 consecutive acidic amino acids selected from,aspartates and glutamates. In some embodiments, A has a sequencecomprising 5 to 8 consecutive acidic amino acids selected from,aspartates and glutamates. In some embodiments, A has a sequencecomprising 5 to 7 consecutive acidic amino acids selected from,aspartates and glutamates. In some embodiments, A has a sequencecomprising 5 consecutive acidic amino acids selected from, aspartatesand glutamates. In some embodiments, A has a sequence comprising 6consecutive acidic amino acids selected from, aspartates and glutamates.In some embodiments, A has a sequence comprising 7 consecutive acidicamino acids selected from, aspartates and glutamates. In someembodiments, A has a sequence comprising 8 consecutive acidic aminoacids selected from, aspartates and glutamates. In some embodiments, Ahas a sequence comprising 9 consecutive acidic amino acids selectedfrom, aspartates and glutamates.

In some embodiments, peptide portion A comprises between about 2 toabout 20 glutamates. In some embodiments, peptide portion A comprisesbetween about 5 to about 20 glutamates. In some embodiments, A has asequence comprising 5 to 9 glutamates. In some embodiments, A has asequence comprising 5 to 8 glutamates. In some embodiments, A has asequence comprising 5 to 7 glutamates. In some embodiments, A has asequence comprising 5 glutamates. In some embodiments, A has a sequencecomprising 6 glutamates. In some embodiments, A has a sequencecomprising 7 glutamates. In some embodiments, A has a sequencecomprising 8 glutamates. In some embodiments, A has a sequencecomprising 9 glutamates.

In some embodiments, peptide portion A comprises between about 2 toabout 20 consecutive glutamates. In some embodiments, peptide portion Acomprises between about 5 to about 20 consecutive glutamates. In someembodiments, A has a sequence comprising 5 to 9 consecutive glutamates(SEQ ID NO: 13). In some embodiments, A has a sequence comprising 5 to 8consecutive glutamates (SEQ ID NO: 14). In some embodiments, A has asequence comprising 5 to 7 consecutive glutamates (SEQ ID NO: 15). Insome embodiments, A has a sequence comprising 5 consecutive glutamates(SEQ ID NO: 4). In some embodiments, A has a sequence comprising 6consecutive glutamates (SEQ ID NO: 16). In some embodiments, A has asequence comprising 7 consecutive glutamates (SEQ ID NO: 17). In someembodiments, A has a sequence comprising 8 consecutive glutamates (SEQID NO: 18). In some embodiments, A has a sequence comprising 9consecutive glutamates (SEQ ID NO: 5).

In some embodiments, portion A comprises 5 consecutive glutamates (i.e.,EEEEE (SEQ ID NO: 4) or eeeee). In some embodiments, portion A comprises9 consecutive glutamates (i.e., EEEEEEEEE (SEQ ID NO: 5) or eeeeeeeee).

An acidic portion A may include amino acids that are not acidic. Acidicportion A may comprise other moieties, such as negatively chargedmoieties. In embodiments of a selective delivery molecule disclosedherein, an acidic portion A may be a negatively charged portion,preferably having about 2 to about 20 negative charges at physiologicalpH that does not include an amino acid.

In some embodiments, the amount of negative charge in portion A isapproximately the same as the amount of positive charge in portion B. Insome embodiments, the amount of negative charge in portion A is not thesame as the amount of positive charge in portion B. In some embodiments,improved tissue uptake is seen in a selective delivery molecule whereinthe amount of negative charge in portion A is not the same as the amountof positive charge in portion B. In some embodiments, improvedsolubility is observed in a selective delivery molecule wherein theamount of negative charge in portion A is not the same as the amount ofpositive charge in portion B. In some embodiments, faster tissue uptakeis seen in a selective delivery molecule wherein the amount of negativecharge in portion A is not the same as the amount of positive charge inportion B. In some embodiments, greater tissue uptake is seen in aselective delivery molecule wherein the amount of negative charge inportion A is not the same as the amount of positive charge in portion B.

Portion A is either L-amino acids or D-amino acids. In embodiments ofthe invention, D-amino acids are preferred in order to minimizeimmunogenicity and nonspecific cleavage by background peptidases orproteases. Cellular uptake of oligo-D-arginine sequences is known to beas good as or better than that of oligo-L-arginines.

It will be understood that portion A may include non-standard aminoacids, such as, for example, hydroxylysine, desmosine, isodesmosine, orother non-standard amino acids. Portion A may include modified aminoacids, including post-translationally modified amino acids such as, forexample, methylated amino acids (e.g., methyl histidine, methylatedforms of lysine, etc.), acetylated amino acids, amidated amino acids,formylated amino acids, hydroxylated amino acids, phosphorylated aminoacids, or other modified amino acids. Portion A may also include peptidemimetic moieties, including portions linked by non-peptide bonds andamino acids linked by or to non-amino acid portions.

The Selective Delivery Molecules disclosed herein are effective where Ais at the amino terminus or where A is at the carboxy terminus, i.e.,either orientation of the peptide bonds is permissible.

Portion B

In some embodiments, B is a peptide with a sequence comprising 5 to 15basic amino acids. In some embodiments, peptide portion B comprisesbetween about 5 to about 20 basic amino acids. In some embodiments,peptide portion B comprises between about 5 to about 12 basic aminoacids. In some embodiments, peptide portion B comprises between about 7to about 9 basic amino acids. In some embodiments, peptide portion Bcomprises between about 7 to about 8 basic amino acids. In someembodiments, peptide portion B comprises 9 basic amino acids. In someembodiments, peptide portion B comprises 8 basic amino acids. In someembodiments, peptide portion B comprises 7 basic amino acids.

In some embodiments, peptide portion B comprises between about 5 toabout 20 consecutive basic amino acids. In some embodiments, peptideportion B comprises between about 5 to about 12 consecutive basic aminoacids. In some embodiments, peptide portion B comprises between about 7to about 9 consecutive basic amino acids. In some embodiments, peptideportion B comprises between about 7 to about 8 consecutive basic aminoacids. In some embodiments, peptide portion B comprises 9 consecutivebasic amino acids. In some embodiments, peptide portion B comprises 8consecutive basic amino acids. In some embodiments, peptide portion Bcomprises 7 consecutive basic amino acids.

In some embodiments, peptide portion B comprises between about 5 toabout 20 basic amino acids selected from arginines, histidines, andlysines. In some embodiments, peptide portion B comprises between about5 to about 12 basic amino acids selected from arginines, histidines, andlysines. In some embodiments, peptide portion B comprises between about7 to about 9 basic amino acids selected from arginines, histidines, andlysines. In some embodiments, peptide portion B comprises between about7 to about 8 basic amino acids selected from arginines, histidines, andlysines. In some embodiments, peptide portion B comprises 9 basic aminoacids selected from arginines, histidines, and lysines. In someembodiments, peptide portion B comprises 8 basic amino acids selectedfrom arginines, histidines, and lysines. In some embodiments, peptideportion B comprises 7 basic amino acids selected from arginines,histidines, and lysines.

In some embodiments, peptide portion B comprises between about 5 toabout 20 consecutive basic amino acids selected from arginines,histidines, and lysines. In some embodiments, peptide portion Bcomprises between about 5 to about 12 consecutive basic amino acidsselected from arginines, histidines, and lysines. In some embodiments,peptide portion B comprises between about 7 to about 9 consecutive basicamino acids selected from arginines, histidines, and lysines. In someembodiments, peptide portion B comprises between about 7 to about 8consecutive basic amino acids selected from arginines, histidines, andlysines. In some embodiments, peptide portion B comprises 9 consecutivebasic amino acids selected from arginines, histidines, and lysines. Insome embodiments, peptide portion B comprises 8 consecutive basic aminoacids selected from arginines, histidines, and lysines. In someembodiments, peptide portion B comprises 7 consecutive basic amino acidsselected from arginines, histidines, and lysines.

In some embodiments, peptide portion B comprises between about 5 toabout 20 arginines. In some embodiments, peptide portion B comprisesbetween about 5 to about 12 arginines. In some embodiments, peptideportion B comprises between about 7 to about 9 arginines. In someembodiments, peptide portion B comprises between about 7 to about 8arginines. In some embodiments, peptide portion B comprises 9 arginines.In some embodiments, peptide portion B comprises 8 arginines. In someembodiments, peptide portion B comprises 7 arginines.

In some embodiments, peptide portion B comprises between about 5 toabout 20 consecutive arginines. In some embodiments, peptide portion Bcomprises between about 5 to about 12 consecutive arginines. In someembodiments, peptide portion B comprises between about 7 to about 9consecutive arginines. In some embodiments, peptide portion B comprisesbetween about 7 to about 8 consecutive arginines. In some embodiments,peptide portion B comprises 9 consecutive arginines (SEQ ID NO: 7). Insome embodiments, peptide portion B comprises 8 consecutive arginines(SEQ ID NO: 6). In some embodiments, peptide portion B comprises 7consecutive arginines (SEQ ID NO: 19).

A basic portion B may include amino acids that are not basic. Basicportion B may comprise other moieties, such as positively chargedmoieties. In embodiments, a basic portion B may be a positively chargedportion, preferably having between about 5 and about 20 positive chargesat physiological pH, that does not include an amino acid. In someembodiments, the amount of negative charge in portion A is approximatelythe same as the amount of positive charge in portion B. In someembodiments, the amount of negative charge in portion A is not the sameas the amount of positive charge in portion B.

Portion B is either L-amino acids or D-amino acids. In embodiments ofthe invention, D-amino acids are preferred in order to minimizeimmunogenicity and nonspecific cleavage by background peptidases orproteases. Cellular uptake of oligo-D-arginine sequences is known to beas good as or better than that of oligo-L-arginines.

It will be understood that portion B may include non-standard aminoacids, such as, for example, hydroxylysine, desmosine, isodesmosine, orother non-standard amino acids. Portion B may include modified aminoacids, including post-translationally modified amino acids such as, forexample, methylated amino acids (e.g., methyl histidine, methylatedforms of lysine, etc.), acetylated amino acids, amidated amino acids,formylated amino acids, hydroxylated amino acids, phosphorylated aminoacids, or other modified amino acids. Portion B may also include peptidemimetic moieties, including portions linked by non-peptide bonds andamino acids linked by or to non-amino acid portions.

In embodiments where X is a peptide cleavable by a protease, it may bepreferable to join the C-terminus of X to the N-terminus of B, so thatthe new amino terminus created by cleavage of X contributes anadditional positive charge that adds to the positive charges alreadypresent in B.

Conjugation Group (c)

In some embodiments, the cargo (e.g., D_(A) and D_(B)) and themacromolecule carriers (M) are attached indirectly to A-X-B.

In some embodiments, the cargo (e.g., D_(A) and D_(B)) and themacromolecule carriers (M) are attached indirectly to A-X-B by aconjugation group (c_(A), c_(B), and c_(M)). In some embodiments, thecargo (e.g., D_(A) and D_(B)) and the macromolecule carriers (M) areattached indirectly to A-X-B by a reactive conjugation group (c_(A),c_(B), and c_(M)). In some embodiments, the cargo (e.g., D_(A) andD_(B)) and the macromolecule carriers (M) are attached indirectly toA-X-B by an orthogonally reactive conjugation group (c_(A), c_(B), andc_(M)). In some embodiments, c_(A), c_(B), and c_(M) are independentlyan amino acid. In some embodiments, c_(A), c_(B), and c_(M) areindependently 0-10 amino acids. In some embodiments, c_(A), c_(B), andc_(M) are independently 1 amino acid. In some embodiments, c_(A), c_(B),and c_(M) are independently 2 amino acids. In some embodiments, c_(A),c_(B), and c_(M) are independently 3 amino acids. In some embodiments,c_(A), c_(B), and c_(M) are independently 4 amino acids. In someembodiments, c_(A), c_(B), and c_(M) are independently 5 amino acids. Insome embodiments, c_(A), c_(B), and c_(M) are independently 6 aminoacids. In some embodiments, c_(A), c_(B), and c_(M) are independently 7amino acids. In some embodiments, c_(A), c_(B), and c_(M) areindependently 8 amino acids. In some embodiments, c_(A), c_(B), andc_(M) are independently 9 amino acids. In some embodiments, c_(A),c_(B), and c_(M) are independently 10 amino acids.

In some embodiments, c_(A), c_(B), and c_(M) are independently aderivatized amino acid. In some embodiments, multiple cargos (D) areattached to a derivatized amino acid conjugation group.

In some embodiments, the conjugation group comprises a receptor ligand.

In some embodiments, c_(A), c_(B), and c_(M) each independently comprisea naturally-occurring amino acid or a non-naturally-occurring aminoacid. In some embodiments, c_(A), c_(B), and c_(M) each independentlycomprise a D amino acid, a L amino acid, an α-amino acid, a β-aminoacid, or a δ-amino acid. In some embodiments, c_(A), c_(B), and c_(M)each independently comprise any amino acid having a free thiol group,any amino acid containing a free amine group, any amino acid having aN-terminal amine group, and any amino acid with a side chain capable offorming an oxime or hydrazone bond upon reaction with a hydroxylamine orhydrazine group. In some embodiments, c_(A), c_(B), and c_(M) eachindependently comprise D-cysteine, D-glutamate, lysine, andpara-4-acetyl L-phenylalanine. In some embodiments, c_(B) comprises anyamino acid having a free thiol group. In some embodiments, c_(B)comprises D-cysteine. In some embodiments, c_(A) comprises any aminoacid having a N-terminal amine group. In some embodiments, c_(A)comprises D-glutamate. In some embodiments, c_(A) comprises lysine. Insome embodiments, c_(M) comprises any amino acid with a side chaincapable of forming an oxime or hydrazone bond upon reaction with ahydroxylamine or hydrazine group. In some embodiments, c_(M) comprisespara-4-acetyl L-phenylalanine.

In some embodiments, c_(A), c_(B), and c_(M) are each independentlyselected from a naturally-occurring amino acid or anon-naturally-occurring amino acid. In some embodiments, c_(A), c_(B),and c_(M) are each independently selected from a D amino acid, a L aminoacid, an α-amino acid, a β-amino acid, and a δ-amino acid. In someembodiments, c_(A), c_(B), and c_(M) are each independently any aminoacid having a free thiol group, any amino acid containing a free aminegroup, any amino acid having a N-terminal amine group, and any aminoacid with a side chain capable of forming an oxime or hydrazone bondupon reaction with a hydroxylamine or hydrazine group. In someembodiments, c_(A), c_(B), and c_(M) are each independently selectedfrom: D-cysteine, D-glutamate, lysine, and para-4-acetylL-phenylalanine. In some embodiments, c_(B) is any amino acid having afree thiol group. In some embodiments, c_(B) is D-cysteine. In someembodiments, c_(A) is any amino acid having a N-terminal amine group. Insome embodiments, c_(A) is D-glutamate. In some embodiments, c_(A) islysine. In some embodiments, c_(M) is any amino acid with a side chaincapable of forming an oxime or hydrazone bond upon reaction with ahydroxylamine or hydrazine group. In some embodiments, c_(M) ispara-4-acetyl L-phenylalanine.

Cargo (D) Imaging Agents

In some embodiments, an imaging agent is a dye. In some embodiments, animaging agent is a fluorescent moiety. In some embodiments, afluorescent moiety is selected from: a fluorescent protein, afluorescent peptide, a fluorescent dye, a fluorescent material or acombination thereof.

All fluorescent moieties are encompassed within the term “fluorescentmoiety.” Specific examples of fluorescent moieties given herein areillustrative and are not meant to limit the fluorescent moieties for usewith the targeting molecules disclosed herein.

Examples of fluorescent dyes include, but are not limited to, xanthenes(e.g., rhodamines, rhodols and fluoresceins, and their derivatives);bimanes; coumarins and their derivatives (e.g., umbelliferone andaminomethyl coumarins); aromatic amines (e.g., dansyl; squarate dyes);benzofurans; fluorescent cyanines; indocarbocyanines; carbazoles;dicyanomethylene pyranes; polymethine; oxabenzanthrane; xanthene;pyrylium; carbostyl; perylene; acridone; quinacridone; rubrene;anthracene; coronene; phenanthrecene; pyrene; butadiene; stilbene;porphyrin; pthalocyanine; lanthanide metal chelate complexes; rare-earthmetal chelate complexes; and derivatives of such dyes.

Examples of fluorescein dyes include, but are not limited to,5-carboxyfluorescein, fluorescein-5-isothiocyanate,fluorescein-6-isothiocyanate and 6-carboxyfluorescein.

Examples of rhodamine dyes include, but are not limited to,tetramethylrhodamine-6-isothiocyanate, 5-carboxytetramethylrhodamine,5-carboxy rhodol derivatives, tetramethyl and tetraethyl rhodamine,diphenyldimethyl and diphenyldiethyl rhodamine, dinaphthyl rhodamine,rhodamine 101 sulfonyl chloride (sold under the tradename of TEXASRED®).

Examples of cyanine dyes include, but are not limited to, Cy3, Cy3B,Cy3.5, Cy5, Cy5.5, Cy7, IRDYE680, Alexa Fluor 750, IRDye800CW, ICG.

Examples of fluorescent peptides include GFP (Green Fluorescent Protein)or derivatives of GFP (e.g., EBFP, EBFP2, Azurite, mKalamal, ECFP,Cerulean, CyPet, YFP, Citrine, Venus, YPet).

Fluorescent labels are detected by any suitable method. For example, afluorescent label may be detected by exciting the fluorochrome with theappropriate wavelength of light and detecting the resultingfluorescence, e.g., by microscopy, visual inspection, via photographicfilm, by the use of electronic detectors such as charge coupled devices(CCDs), photomultipliers, etc.

In some embodiments, the imaging agent is labeled with apositron-emitting isotope (e.g., ¹⁸F) for positron emission tomography(PET), gamma-ray isotope (e.g., ^(99m)Tc) for single photon emissioncomputed tomography (SPECT), or a paramagnetic molecule or nanoparticle(e.g., Gd³⁺ chelate or coated magnetite nanoparticle) for magneticresonance imaging (MRI).

In some embodiments, the imaging agent is labeled with: a gadoliniumchelate, an iron oxide particle, a super paramagnetic iron oxideparticle, an ultra small paramagnetic particle, a manganese chelate orgallium containing agent.

Examples of gadolinium chelates include, but are not limited todiethylene triamine pentaacetic acid (DTPA),1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and1,4,7-triazacyclononane-N,N′,N″-triacetic acid (NOTA).

In some embodiments, the imaging agent is a near-infrared fluorophorefor near-infra red (near-IR) imaging, a luciferase (firefly, bacterial,or coelenterate) or other luminescent molecule for bioluminescenceimaging, or a perfluorocarbon-filled vesicle for ultrasound.

In some embodiments, the imaging agent is a nuclear probe. In someembodiments, the imaging agent is a SPECT or PET radionuclide probe. Insome embodiments, the radionuclide probe is selected from: a technetiumchelate, a copper chelate, a radioactive fluorine, a radioactive iodine,a indiuim chelate.

Examples of Tc chelates include, but are not limited to HYNIC, DTPA, andDOTA.

In some embodiments, the imaging agent contains a radioactive moiety,for example a radioactive isotope such as ²¹¹At, ¹³¹I, ¹²⁵I, ⁹⁰Y, ¹⁸⁶Re,¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, ³²P, ⁶⁴Cu radioactive isotopes of Lu, and others.

In some embodiments, a selective delivery molecule according to FormulaI comprising an imaging agent is employed in guided surgery. In someembodiments, the selective delivery molecule preferentially localized tocancerous, or other undesirable tissues (i.e. necrotic tissues). In someembodiments, a selective delivery molecule according to Formula Icomprising an imaging agent is employed in a guided surgery to removecolorectal cancer. In some embodiments, guided surgery employing theselective delivery molecule allows a surgeon to excise as little healthy(i.e., non-cancerous) tissue as possible. In some embodiments, guidedsurgery employing the selective delivery molecule allows a surgeon tovisualize and excise more cancerous tissue than the surgeon would havebeen able to excise without the presence of the selective deliverymolecule. In some embodiments, the surgery is fluorescence-guidedsurgery.

Macromolecular Carriers (M)

The term “carrier” means an inert molecule that modulates plasmahalf-life, solubility, or bio-distribution. In some embodiments, acarrier modulates plasma half-life of a selective delivery moleculedisclosed herein. In some embodiments, a carrier modulates solubility ofa selective delivery molecule disclosed herein. In some embodiments, acarrier modulates bio-distribution of a selective delivery moleculedisclosed herein.

In some embodiments, a carrier decreases uptake of a selective deliverymolecule by non-target cells or tissues. In some embodiments, a carrierdecreases uptake of a selective delivery molecule into cartilage. Insome embodiments, a carrier decreases uptake of a selective deliverymolecule into joints relative to target tissue.

In some embodiments, a carrier increases uptake of a selective deliverymolecule by target cells or tissues. In some embodiments, a carrierdecreases uptake of a selective delivery molecule into the liverrelative to target tissue. In some embodiments, a carrier decreasesuptake of a selective delivery molecule into kidneys. In someembodiments, a carrier enhances uptake into cancer tissue. In someembodiments, a carrier enhances uptake into lymphatic channels and/orlymph nodes.

In some embodiments, a carrier increases plasma half-life by reducingglomerular filtration. In some embodiments, a carrier modulates plasmahalf-life by increasing or decreases metabolism or protease degradation.In some embodiments, a carrier increases tumor uptake due to enhancedpermeability and retention (EPR) of tumor vasculature. In someembodiments, a carrier increases the aqueous solubility of selectivedelivery molecule.

In some embodiments, any M is independently directly or indirectly(e.g., via c_(M)) bound to A, B, or X. In some embodiments, any M isindependently bound to A at the n-terminal poly glutamate. In someembodiments, any M is independently bound to A (or, the n-terminal polyglutamate) by a covalent linkage. In some embodiments, any M isindependently bound to B at the c-terminal polyarginine. In someembodiments, any M is independently bound to B (or, the c-terminalpolyarginine) by a covalent linkage. In some embodiments, any M isindependently directly or indirectly bound to linkers between X and A, Xand B, B and C/N terminus, and A and C/N terminus. In some embodiments,the covalent linkage comprises an ether bond, thioether bond, aminebond, amide bond, oxime bond, carbon-carbon bond, carbon-nitrogen bond,carbon-oxygen bond, or carbon-sulfur bond.

In some embodiments, M is selected from a protein, a synthetic ornatural polymer, or a dendrimer. In some embodiments, M is selected fromdextran, a PEG polymer (e.g., PEG 0.5 kDa, PEG 2 kDa, PEG 5 kDa, PEG 12kDa, PEG 20 kDa, PEG 30 kDa, and PEG40 kDa), albumin, or a combinationthereof. In some embodiments, M is a PEG polymer.

In some embodiments, the size of M is between 50 and 70 kD.

In some embodiments, the selective delivery molecule is conjugated toalbumin. In certain instances, albumin is excluded from the glomerularfiltrate under normal physiological conditions. In some embodiments, theselective delivery molecule comprises a reactive group such as maleimidethat can form a covalent conjugate with albumin. A selective deliverymolecule comprising albumin results in enhanced accumulation of cleavedselective delivery molecules in tumors in a cleavage dependent manner.In some embodiments, albumin conjugates have good pharmacokineticproperties.

In some embodiments, the selective delivery molecule is conjugated toPEG polymers. In some embodiments, the selective delivery molecule isconjugated to PEG polymers having an average molecular weight ofapproximately 0.5 kDa (PEG 0.5 kDa). In some embodiments, the selectivedelivery molecule is conjugated to PEG polymers having an averagemolecular weight of approximately 1 kDa (PEG 1 kDa). In someembodiments, the selective delivery molecule is conjugated to PEGpolymers having an average molecular weight of approximately 2 kDa (PEG2 kDa). In some embodiments, the selective delivery molecule isconjugated to PEG polymers having an average molecular weight ofapproximately 5 kDa (PEG 5 kDa). In some embodiments, the selectivedelivery molecule is conjugated to PEG polymers having an averagemolecular weight of approximately 10 kDa (PEG 10 kDa). In someembodiments, the selective delivery molecule is conjugated PEG polymershaving an average molecular weight of approximately 12 kDa (PEG 12 kDa).In some embodiments, selective delivery molecule is conjugated to PEGpolymers having an average molecular weight of approximately 20 kDa (PEG20 kDa). In some embodiments, selective delivery molecule is conjugatedto PEG polymers having an average molecular weight of approximately 30kDa (PEG 30 kDa). In some embodiments, selective delivery moleculesconjugated to PEG30 kDa had a longer half-life as compared to freepeptides. In some embodiments, selective delivery molecules areconjugated to PEG polymers having an average molecular weight of betweenabout 20 to about 40 kDa which have hepatic and renal clearance.

In some embodiments, the selective delivery molecule is conjugated to adextran. In some embodiments, the selective delivery molecule isconjugated to a 70 kDa dextran. In some embodiments, dextran conjugates,being a mixture of molecular weights, are difficult to synthesize andpurify reproducibly.

In some embodiments, the selective delivery molecule is conjugated tostreptavidin.

In some embodiments, the selective delivery molecule is conjugated to afifth generation PAMAM dendrimer.

In some embodiments, a carrier is capped. In some embodiments, capping acarrier improves the pharmacokinetics and reduces cytotoxicity of acarrier by adding hydrophilicity. In some embodiments, the cap isselected from: Acetyl, succinyl, 3-hydroxypropionyl, 2-sulfobenzoyl,glycidyl, PEG-2 kDa, PEG-4 kDa, PEG-8 kDa and PEG-12 kDa.

Portion X (Linkers)

In some embodiments, a linker consisting of one or more amino acids isused to join peptide sequence A (i.e., the sequence designed to inhibitthe delivery action of peptide B) and peptide sequence B. Generally thepeptide linker will have no specific biological activity other than tojoin the molecules or to preserve some minimum distance or other spatialrelationship between them. However, the constituent amino acids of thelinker may be selected to influence some property of the molecule suchas the folding, net charge, or hydrophobicity.

In live cells, an intact selective delivery molecule disclosed hereinmay not be able to enter the cell because of the presence of portion A.Thus, a strictly intracellular process for cleaving X would beineffective to cleave X in healthy cells since portion A, preventinguptake into cells, would not be effectively cleaved by intracellularenzymes in healthy cells since it would not be taken up and would notgain access to such intracellular enzymes. However, where a cell isinjured or diseased (e.g., cancerous cells, hypoxic cells, ischemiccells, apoptotic cells, necrotic cells) such intracellular enzymes leakout of the cell and cleavage of A would occur, allowing entry of portionB and/or cargo into the cell, effecting targeted delivery of portion Band/or cargo D to neighboring cells. In some embodiments, X is cleavedin the extracellular space.

In certain instances, capillaries are leaky around tumors and othertrauma sites. In some embodiments, leaky capillaries enhance the abilityof high molecular weight molecules (e.g., molecular weight of about 30kDa or more) to reach the interstitial compartment. In some embodiments,X linker is cleaved adjacent to cancerous tissue. In some embodiments,cells that do not express the relevant protease but that are immediatelyadjacent to cells expressing the relevant protease pick up cargo from aselective delivery molecule because linkage of a X linker is typicallyextracellular. In some embodiments, such bystander targeting isbeneficial in the treatment of tumors because of the heterogeneity ofcell phenotypes and the wish to eliminate as high a percentage ofsuspicious cells as possible.

In some embodiments, X is a cleavable linker.

In some embodiments, the linker is flexible. In some embodiments, thelinker is rigid.

In some embodiments, the linker comprises a linear structure. In someembodiments, the linker comprises a non-linear structure. In someembodiments, the linker comprises a branched structure. In someembodiments, the linker comprises a cyclic structure.

In some embodiments, X is about 5 to about 30 atoms in length. In someembodiments, X is about 6 atoms in length. In some embodiments, X isabout 8 atoms in length. In some embodiments, X is about 10 atoms inlength. In some embodiments, X is about 12 atoms in length. In someembodiments, X is about 14 atoms in length. In some embodiments, X isabout 16 atoms in length. In some embodiments, X is about 18 atoms inlength. In some embodiments, X is about 20 atoms in length. In someembodiments, X is about 25 atoms in length. In some embodiments, X isabout 30 atoms in length.

In some embodiments, the linker binds peptide portion A (i.e., thepeptide sequence which prevents cellular uptake) to peptide portion B(i.e., the delivery sequence) by a covalent linkage. In someembodiments, the covalent linkage comprises an ether bond, thioetherbond, amine bond, amide bond, oxime bond, hydrazone bond, carbon-carbonbond, carbon-nitrogen bond, carbon-oxygen bond, or carbon-sulfur bond.

In some embodiments, X comprises a peptide linkage. The peptide linkagecomprises L-amino acids and/or D-amino acids. In embodiments of theinvention, D-amino acids are preferred in order to minimizeimmunogenicity and nonspecific cleavage by background peptidases orproteases. Cellular uptake of oligo-D-arginine sequences is known to beas good as or better than that of oligo-L-arginines.

In some embodiments, a X linker is designed for cleavage in the presenceof particular conditions or in a particular environment. In preferredembodiments, a X linker is cleavable under physiological conditions.Cleavage of such a X linker may, for example, be enhanced or may beaffected by particular pathological signals or a particular environmentrelated to cells in which cargo delivery is desired. The design of a Xlinker for cleavage by specific conditions, such as by a specificenzyme, allows the targeting of cellular uptake to a specific locationwhere such conditions obtain. Thus, one important way that selectivedelivery molecules provide specific targeting of cellular uptake todesired cells, tissues, or regions is by the design of the linkerportion X to be cleaved by conditions near such targeted cells, tissues,or regions.

In some embodiments, X is a pH-sensitive linker. In some embodiments, Xis cleaved under basic pH conditions. In some embodiments, X is cleavedunder acidic pH conditions. In some embodiments, X is cleaved by aprotease, a matrix metalloproteinase, or a combination thereof. In someembodiments, X is cleaved by a reducing agent. In some embodiments X iscleaved by an oxidizing agent.

In some embodiments, X is cleaved by an MMP. The hydrolytic activity ofmatrix metalloproteinases (MMPs) has been implicated in the invasivemigration of metastatic tumor cells. In certain instances, MMPs arefound near sites of inflammation. In certain instances, MMPs are foundnear sites of stroke (i.e., a disorder characterized by brain damagefollowing a decrease in blood flow). Thus, uptake of molecules havingfeatures of the invention are able to direct cellular uptake of cargo(at least one D moiety) to specific cells, tissues, or regions havingactive MMPs in the extracellular environment. In some embodiments, Xcomprises an amino-acid sequence selected from: PLG-C(Me)-AG (SEQ ID NO:1), PLGLAG (SEQ ID NO: 2) or RPLALWRS (SEQ ID NO: 3). In someembodiments, X is cleaved by a metalloproteinase enzymes selected fromMMP-2, MMP-9, or MMP-7 (MMPs involved in cancer and inflammation). Insome embodiments, the linker is cleaved by MMP-2. In some embodiments,the linker is cleaved by MMP-9. In some embodiments, the linker iscleaved by MMP-7.

In some embodiments, X is cleaved by proteolytic enzymes or reducingenvironment, as may be found near cancerous cells. Such an environment,or such enzymes, are typically not found near normal cells.

In some embodiments, X is cleaved by serine proteases including but notlimited to thrombin and cathepsins. In some embodiments, X is cleaved bycathepsin K, cathepsin S, cathepsin D, cathepsin E, cathepsin W,cathepsin F, cathepsin A, cathepsin C, cathepsin H, cathepsin Z, or anycombinations thereof. In some embodiments, X is cleaved by cathepsin Kand/or cathepsin S.

In some embodiments, X is cleaved in or near tissues suffering fromhypoxia. In some embodiments, cleavage in or near hypoxic tissuesenables targeting of cancer cells and cancerous tissues, infarctregions, and other hypoxic regions. In some embodiments, X comprises adisulfide bond. In some embodiments, a linker comprising a disulfidebond is preferentially cleaved in hypoxic regions and so targets cargodelivery to cells in such a region. Hypoxia is thought to cause cancercells to become more resistant to radiation and chemotherapy, and alsoto initiate angiogenesis. In a hypoxic environment in the presence of,for example, leaky or necrotic cells, free thiols and other reducingagents become available extracellularly, while the O₂ that normallykeeps the extracellular environment oxidizing is by definition depleted.In some embodiments, this shift in the redox balance promotes reductionand cleavage of a disulfide bond within a X linker. In addition todisulfide linkages which take advantage of thiol-disulfide equilibria,linkages including quinones that fall apart when reduced tohydroquinones are used in a X linker designed to be cleaved in a hypoxicenvironment.

In some embodiments, X is cleaved in a necrotic environment. Necrosisoften leads to the release of enzymes or other cell contents that may beused to trigger cleavage of a X linker. In some embodiments, cleavage ofX by necrotic enzymes (e.g., by calpains) allows cargo to be taken up bydiseased cells and by neighboring cells that had not yet become fullyleaky.

In some embodiments, X is an acid-labile linker. In some embodiments, Xcomprises an acetal or vinyl ether linkage. Acidosis is observed insites of damaged or hypoxic tissue, due to the Warburg shift fromoxidative phosphorylation to anaerobic glycolysis and lactic acidproduction. In some embodiments, acidosis is used as a trigger of cargouptake by replacing some of the arginines within B by histidines, whichonly become cationic below pH 7.

It will be understood that a linker disclosed herein may includenon-standard amino acids, such as, for example, hydroxylysine,desmosine, isodesmosine, or other non-standard amino acids. A linkerdisclosed herein may include modified amino acids, includingpost-translationally modified amino acids such as, for example,methylated amino acids (e.g., methyl histidine, methylated forms oflysine, etc.), acetylated amino acids, amidated amino acids, formylatedamino acids, hydroxylated amino acids, phosphorylated amino acids, orother modified amino acids. A linker disclosed herein may also includepeptide mimetic moieties, including portions linked by non-peptide bondsand amino acids linked by or to non-amino acid portions.

In some embodiments, the linker X comprises an amino acid sequenceselected from: PLGLAG (SEQ ID NO: 2), PLG-C(me)-AG (SEQ ID NO: 1),RPLALWRS (SEQ ID NO: 3), ESPAYYTA (SEQ ID NO: 8), DPRSFL (SEQ ID NO: 9),PPRSFL (SEQ ID NO:10), RLQLKL (SEQ ID NO: 11), and RLQLK(Ac) (SEQ ID NO:12). In some embodiments, the linker X comprises the amino acid sequencePLGLAG (SEQ ID NO: 2). In some embodiments, the linker X comprises theamino acid sequence PLG-C(me)-AG (SEQ ID NO: 1). In some embodiments,the linker X comprises the amino acid sequence PLGxAG (SEQ ID NO: 20),wherein x is any amino acid (naturally-occurring or non-naturallyoccurring). In some embodiments, the linker X comprises the amino acidsequence RPLALWRS (SEQ ID NO: 3). In some embodiments, the linker Xcomprises the amino acid sequence ESPAYYTA (SEQ ID NO: 8). In someembodiments, the linker X comprises the amino acid sequence DPRSFL (SEQID NO: 9). In some embodiments, the linker X comprises the amino acidsequence PPRSFL (SEQ ID NO: 10). In some embodiments, the linker Xcomprises the amino acid sequence RLQLKL (SEQ ID NO: 11). In someembodiments, the linker X comprises the amino acid sequence RLQLK(Ac)(SEQ ID NO: 12).

In some embodiments, the linker X comprises a peptide selected from:PR(S/T)(L/I)(S/T), where the letters in parentheses indicate that eitherone of the indicated amino acids may be at that position in thesequence); GGAANLVRGG (SEQ ID NO: 21); SGRIGFLRTA (SEQ ID NO: 22); SGRSA(SEQ ID NO: 23); GFLG (SEQ ID NO:24); ALAL (SEQ ID NO: 25); FK;PIC(Et)F-F (SEQ ID NO: 26), where C(Et) indicates S-ethylcysteine (acysteine with an ethyl group attached to the thiol) and the “-”indicates the typical cleavage site in this and subsequent sequences);GGPRGLPG (SEQ ID NO: 27); HSSKLQ (SEQ ID NO: 28); LVLA-SSSFGY (SEQ IDNO: 29); GVSQNY-PIVG (SEQ ID NO: 30); GVVQA-SCRLA (SEQ ID NO: 31);f(Pip)R-S, where “f” indicates D-phenylalanine and “Pip” indicatespiperidine-2-carboxylic acid (pipecolinic acid, a proline analog havinga six-membered ring); DEVD (SEQ ID NO: 32); GWEHDG (SEQ ID NO: 33);RPLALWRS (SEQ ID NO: 3), or a combination thereof.

In some embodiments, X is cleaved under hypoxic conditions. In someembodiments, X comprises a disulfide linkage. In some embodiments, Xcomprises a quinine.

In some embodiments, X is cleaved under necrotic conditions. In someembodiments, X comprises a molecule cleavable by a calpain.

In some embodiments, X comprises 6-aminohexanoyl,5-(amino)-3-oxapentanoyl, or a combination thereof. In some embodiments,X comprises a disulfide linkage.

In some embodiments, the linker is an alkylene. In some embodiments, thelinker is an alkenylene. In some embodiments, the linker is analkynylene. In some embodiments, the linker is a heteroalkylene.

An “alkylene” group refers to an aliphatic hydrocarbon group. Thealkylene moiety is a diradical and may be a saturated alkylene or anunsaturated alkylene.

The “alkylene” moiety may have 1 to 10 carbon atoms (whenever it appearsherein, a numerical range such as “1 to 10” refers to each integer inthe given range; e.g., “1 to 10 carbon atoms” means that the alkyl groupmay consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., upto and including 10 carbon atoms, although the present definition alsocovers the occurrence of the term “alkylene” where no numerical range isdesignated). The alkylene group could also be a “lower alkylene” having1 to 6 carbon atoms. The alkylene group of the compounds describedherein may be designated as “C1-C4 alkylene” or similar designations. Byway of example only, “C1-C4 alkylene” indicates that there are one tofour carbon atoms in the alkylene chain, i.e., the alkylene chain isselected from: methylene, ethylene, propylene, iso-propylene,n-butylene, iso-butylene, sec-butylene, and t-butylene. Typical alkylenegroups include, but are in no way limited to, methylene, ethylene,propylene, isopropylene, butylene, isobutylene, tertiary butylene,pentylene, hexylene, ethenylene, propenylene, butenylene, and the like.

In some embodiments, the linker comprises a diradical ring structure(e.g., an arylene). As used herein, the term “ring” refers to anycovalently closed structure. Rings include, for example, carbocycles(e.g., arylenes and cycloalkylenes), heterocyclenes (e.g.,heteroarylenes and non-aromatic heterocyclenes), aromatics (e.g.arylenes and heteroarylenes), and non-aromatics (e.g., cycloalkylenesand non-aromatic heterocyclenes). Rings can be optionally substituted.Rings can be monocyclic or polycyclic.

As used herein, the term “arylene” refers to a aromatic ring diradicalwherein each of the atoms forming the ring is a carbon atom. Arylenerings can be formed by five, six, seven, eight, nine, or more than ninecarbon atoms. Arylene groups can be optionally substituted. Examples ofarylene groups include, but are not limited to phenylene,naphthalenylene, phenanthrenylene, anthracenylene, fluorenylene, andindenylene.

The term “cycloalkylene” refers to a monocyclic or polycyclicnon-aromatic diradical, wherein each of the atoms forming the ring (i.e.skeletal atoms) is a carbon atom. Cycloalkylenes may be saturated, orpartially unsaturated. Cycloalkylene groups include groups having from 3to 10 ring atoms. Cycloalkylenes include, but are not limited to,cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene,cycloheptylene, and cyclooctylene.

In some embodiments, the ring is a cycloalkane. In some embodiments, thering is a cycloalkene.

In some embodiments, the ring is an aromatic ring. The term “aromatic”refers to a planar ring having a delocalized 7c-electron systemcontaining 4n+2π electrons, where n is an integer. Aromatic rings can beformed from five, six, seven, eight, nine, or more than nine atoms.Aromatics can be optionally substituted. The term “aromatic” includesboth carbocyclic arylene (e.g., phenylene) and heterocyclic arylene (or“heteroarylene” or “heteroaromatic”) groups (e.g., pyridinylene). Theterm includes monocyclic or fused-ring polycyclic (i.e., rings whichshare adjacent pairs of carbon atoms) groups.

In some embodiments, the ring is a heterocyclene. The term“heterocyclene” refers to diradical heteroaromatic and heteroalicyclicgroups containing one to four heteroatoms each selected from O, S and N,wherein each heterocyclic group has from 4 to 10 atoms in its ringsystem, and with the proviso that the ring of said group does notcontain two adjacent 0 or S atoms. Non-aromatic heterocyclic groupsinclude groups having only 3 atoms in their ring system, but aromaticheterocyclic groups must have at least 5 atoms in their ring system. Theheterocyclic groups include benzo-fused ring systems. An example of a3-membered heterocyclic group is aziridinylene. An example of a4-membered heterocyclic group is azetidinylene (derived from azetidine).An example of a 5-membered heterocyclic group is thiazolylene. Anexample of a 6-membered heterocyclic group is pyridylene, and an exampleof a 10-membered heterocyclic group is quinolinylene. Examples ofnon-aromatic heterocyclic groups are pyrrolidinylene,tetrahydrofuranylene, dihydrofuranylene, tetrahydrothienylene,tetrahydropyranylene, dihydropyranylene, tetrahydrothiopyranylene,piperidinylene, morpholinylene, thiomorpholinylene, thioxanylene,piperazinylene, azetidinylene, oxetanylene, thietanylene,homopiperidinylene, oxepanylene, thiepanylene, oxazepinylene,diazepinylene, thiazepinylene, 1,2,3,6-tetrahydropyridinylene,2-pyrrolinylene, 3-pyrrolinylene, indolinylene, 2H-pyranylene,4H-pyranylene, dioxanylene, 1,3-dioxolanylene, pyrazolinylene,dithianylene, dithiolanylene, dihydropyranylene, dihydrothienylene,dihydrofuranylene, pyrazolidinylene, imidazolinylene, imidazolidinylene,3-azabicyclo[3.1.0]hexanylene, 3-azabicyclo[4.1.0]heptanylene,3H-indolylene and quinolizinylene. Examples of aromatic heterocyclicgroups are pyridinylene, imidazolylene, pyrimidinylene, pyrazolylene,triazolylene, pyrazinylene, tetrazolylene, furylene, thienylene,isoxazolylene, thiazolylene, oxazolylene, isothiazolylene, pyrrolylene,quinolinylene, isoquinolinylene, indolylene, benzimidazolylene,benzofuranylene, cinnolinylene, indazolylene, indolizinylene,phthalazinylene, pyridazinylene, triazinylene, isoindolylene,pteridinylene, purinylene, oxadiazolylene, thiadiazolylene,furazanylene, benzofurazanylene, benzothiophenylene, benzothiazolylene,benzoxazolylene, quinazolinylene, quinoxalinylene, naphthyridinylene,and furopyridinylene. The foregoing groups, may be C-attached and/orN-attached where such is possible. The heterocyclic groups includebenzo-fused ring systems and ring systems substituted with one or twooxo (═O) moieties such as pyrrolidin-2-one.

In some embodiments, the ring is fused. The term “fused” refers tostructures in which two or more rings share one or more bonds. In someembodiments, the ring is a dimer. In some embodiments, the ring is atrimer. In some embodiments, the ring is a substituted.

The term “carbocyclic” or “carbocyclene” refers to a diradical ringwherein each of the atoms forming the ring is a carbon atom.Carbocyclene includes arylene and cycloalkylene. The term thusdistinguishes carbocyclene from heterocycene (“heterocyclic”) in whichthe ring backbone contains at least one atom which is different fromcarbon (i.e., a heteroatom). Heterocyclene includes heteroarylene andheterocycloalkylene. Carbocyclenes and heterocyclenes can be optionallysubstituted.

In some embodiments, the linker is substituted. The term “optionallysubstituted” or “substituted” means that the referenced group may besubstituted with one or more additional group(s) individually andindependently selected from C₁-C₆alkyl, C₃-C₈cycloalkyl, aryl,heteroaryl, C₂-C₆heteroalicyclic, hydroxy, C₁-C₆alkoxy, aryloxy,C₁-C₆alkylthio, arylthio, C₁-C₆alkylsulfoxide, arylsulfoxide,C₁-C₆alkylsulfone, arylsulfone, cyano, halo, C₂-C₈acyl, C₂-C₈acyloxy,nitro, C₁-C₆haloalkyl, C₁-C₆fluoroalkyl, and amino, includingC₁-C₆alkylamino, and the protected derivatives thereof. By way ofexample, an optional substituents may be LsRs, wherein each Ls isindependently selected from a bond, —O—, —C(═O)—, —S—, —S(═O)—,—S(═O)₂—, —NH—, —NHC(═O)—, —C(═O)NH—, S(═O)₂NH—, —NHS(═O)₂—, —OC(═O)NH—,—NHC(═O)O—, —(C₁-C₆alkyl)-, or —(C₂-C₆alkenyl)-; and each Rs isindependently selected from H, (C₁-C₄alkyl), (C₃-C₈cycloalkyl),heteroaryl, aryl, and C₁-C₆heteroalkyl. Optionally substitutednon-aromatic groups may be substituted with one or more oxo (═O). Theprotecting groups that may form the protective derivatives of the abovesubstituents are known to those of skill in the art.

In some embodiments, a selective delivery molecules disclosed hereincomprises a single of linker. Use of a single mechanism to mediateuptake of both imaging and therapeutic cargoes is particularly valuable,because imaging with noninjurious tracer quantities can be used to testwhether a subsequent therapeutic dose is likely to concentrate correctlyin the target tissue.

In some embodiments, a selective delivery molecules disclosed hereincomprises a plurality of linkers. Where a selective delivery moleculedisclosed herein includes multiple X linkages, separation of portion Afrom the other portions of the molecule requires cleavage of all Xlinkages. Cleavage of multiple X linkers may be simultaneous orsequential. Multiple X linkages may include X linkages having differentspecificities, so that separation of portion A from the other portionsof the molecule requires that more than one condition or environment(“extracellular signals”) be encountered by the molecule. Cleavage ofmultiple X X linkers thus serves as a detector of combinations of suchextracellular signals. For example, a selective delivery molecule mayinclude two linker portions Xa and Xb connecting basic portion B withacidic portion A. Both Xa and Xb linkers must be cleaved before acidicportion A is separated from basic portion B allowing entry of portion Band cargo moiety C (if any) to enter a cell. It will be understood thata linker region may link to either a basic portion B or a cargo moiety Cindependently of another linker that may be present, and that, wheredesired, more than two linker regions X may be included.

Combinations of two or more X linkers may be used to further modulatethe targeting and delivery of molecules to desired cells, tissue orregions. Combinations of extracellular signals are used to widen ornarrow the specificity of the cleavage of X linkers if desired. Wheremultiple X linkers are linked in parallel, the specificity of cleavageis narrowed, since each X linker must be cleaved before portion A mayseparate from the remainder of the molecule. Where multiple X linkersare linked in series, the specificity of cleavage is broadened, sincecleavage on any one X linker allows separation of portion A from theremainder of the molecule. For example, in order to detect either aprotease OR hypoxia (i.e., to cleave X in the presence of eitherprotease or hypoxia), a X linker is designed to place theprotease-sensitive and reduction-sensitive sites in tandem, so thatcleavage of either would suffice to allow separation of the acidicportion A. Alternatively, in order to detect the presence of both aprotease AND hypoxia (i.e., to cleave X in the presence of both proteaseand hypoxia but not in the presence of only one alone), a X linker isdesigned to place the protease sensitive site between at least one pairof cysteines that are disulfide-bonded to each other. In that case, bothprotease cleavage and disulfide reduction are required in order to allowseparation of portion A.

Exemplary Selective Delivery Molecules

Disclosed herein, in certain embodiments, is a selective deliverymolecule according to SDM-41. Disclosed herein, in certain embodiments,is a selective delivery molecule according to SDM-42. Disclosed herein,in certain embodiments, is a selective delivery molecule according toSDM-43. Disclosed herein, in certain embodiments, is a selectivedelivery molecule according to SDM-44. Disclosed herein, in certainembodiments, is a selective delivery molecule according to SDM-45.Disclosed herein, in certain embodiments, is a selective deliverymolecule according to SDM-46. Disclosed herein, in certain embodiments,is a selective delivery molecule according to SDM-47. Disclosed herein,in certain embodiments, is a selective delivery molecule according toSDM-48. Disclosed herein, in certain embodiments, is a selectivedelivery molecule according to SDM-49. Disclosed herein, in certainembodiments, is a selective delivery molecule according to SDM-50.Disclosed herein, in certain embodiments, is a selective deliverymolecule according to SDM-51. Disclosed herein, in certain embodiments,is a selective delivery molecule according to SDM-52. Disclosed herein,in certain embodiments, is a selective delivery molecule according toSDM-53. Disclosed herein, in certain embodiments, is a selectivedelivery molecule according to SDM-54. Disclosed herein, in certainembodiments, is a selective delivery molecule according to SDM-55.Disclosed herein, in certain embodiments, is a selective deliverymolecule according to SDM-56. Disclosed herein, in certain embodiments,is a selective delivery molecule according to SDM-57. Disclosed herein,in certain embodiments, is a selective delivery molecule according toSDM-58. Disclosed herein, in certain embodiments, is a selectivedelivery molecule according to SDM-59. Disclosed herein, in certainembodiments, is a selective delivery molecule according to SDM-60.Disclosed herein, in certain embodiments, is a selective deliverymolecule according to SDM-61. Disclosed herein, in certain embodiments,is a selective delivery molecule according to SDM-62. Disclosed herein,in certain embodiments, is a selective delivery molecule according toSDM-63. Disclosed herein, in certain embodiments, is a selectivedelivery molecule according to SDM-64. Disclosed herein, in certainembodiments, is a selective delivery molecule according to SDM-65.

The structures of selective delivery molecules SDM-41, SDM-42, SDM-43,SDM-44, SDM-45, SDM-46, SDM-47, SDM-48, SDM-49, SDM-50, SDM-51, SDM-52,SDM-53, SDM-54, SDM-55, SDM-56, SDM-57, SDM-58, SDM-59, SDM-60, SDM-61,SDM-62, SDM-63, SDM-64, and SDM-65 are shown below.

Chemica lStructure SDM-41

SDM-42

SDM-43

SDM-44

SDM-45

SDM-46

SDM-47

SDM-48

SDM-49

SDM-50

SDM-51

SDM-52

SDM-53

SDM-54

SDM-55

SDM-56

SDM-57

SDM-58

SDM-59

SDM-60

SDM-61

SDM-62

SDM-63

SDM-64

SDM-65

Further Modifications

In some embodiments, the targeting molecules of the present inventionare optionally conjugated to high molecular weight molecules thatincrease the multivalency and avidity of labeling. In some embodiments,the high molecular weight molecules are water-soluble polymers. Examplesof suitable water-soluble polymers include, but are not limited to,peptides, saccharides, poly(vinyls), poly(ethers), poly(amines),poly(carboxylic acids) and the like. In some embodiments, thewater-soluble polymer is dextran, polyethylene glycol (PEG),polyoxyalkylene, polysialic acid, starch, or hydroxyethyl starch. Anysuitable method is used to conjugate peptides to water-soluble polymers(see Hermanson G., Bioconjugate Techniques 2^(nd) Ed., Academic Press,Inc. 2008).

Pharmaceutical Compositions

Disclosed herein, in certain embodiments, are pharmaceuticalcompositions comprising a selective delivery molecule according toSDM-41. Disclosed herein, in certain embodiments, are pharmaceuticalcompositions comprising a selective delivery molecule according toSDM-42. Disclosed herein, in certain embodiments, are pharmaceuticalcompositions comprising a selective delivery molecule according toSDM-43. Disclosed herein, in certain embodiments, are pharmaceuticalcompositions comprising a selective delivery molecule according toSDM-44. Disclosed herein, in certain embodiments, are pharmaceuticalcompositions comprising a selective delivery molecule according toSDM-45. Disclosed herein, in certain embodiments, are pharmaceuticalcompositions comprising a selective delivery molecule according toSDM-46. Disclosed herein, in certain embodiments, are pharmaceuticalcompositions comprising a selective delivery molecule according toSDM-47. Disclosed herein, in certain embodiments, are pharmaceuticalcompositions comprising a selective delivery molecule according toSDM-48. Disclosed herein, in certain embodiments, are pharmaceuticalcompositions comprising a selective delivery molecule according toSDM-49. Disclosed herein, in certain embodiments, are pharmaceuticalcompositions comprising a selective delivery molecule according toSDM-50. Disclosed herein, in certain embodiments, are pharmaceuticalcompositions comprising a selective delivery molecule according toSDM-51. Disclosed herein, in certain embodiments, are pharmaceuticalcompositions comprising a selective delivery molecule according toSDM-52. Disclosed herein, in certain embodiments, are pharmaceuticalcompositions comprising a selective delivery molecule according toSDM-53. Disclosed herein, in certain embodiments, are pharmaceuticalcompositions comprising a selective delivery molecule according toSDM-54. Disclosed herein, in certain embodiments, are pharmaceuticalcompositions comprising a selective delivery molecule according toSDM-55. Disclosed herein, in certain embodiments, are pharmaceuticalcompositions comprising a selective delivery molecule according toSDM-56. Disclosed herein, in certain embodiments, are pharmaceuticalcompositions comprising a selective delivery molecule according toSDM-57. Disclosed herein, in certain embodiments, are pharmaceuticalcompositions comprising a selective delivery molecule according toSDM-58. Disclosed herein, in certain embodiments, are pharmaceuticalcompositions comprising a selective delivery molecule according toSDM-59. Disclosed herein, in certain embodiments, are pharmaceuticalcompositions comprising a selective delivery molecule according toSDM-60. Disclosed herein, in certain embodiments, are pharmaceuticalcompositions comprising a selective delivery molecule according toSDM-61. Disclosed herein, in certain embodiments, are pharmaceuticalcompositions comprising a selective delivery molecule according toSDM-62. Disclosed herein, in certain embodiments, are pharmaceuticalcompositions comprising a selective delivery molecule according toSDM-63. Disclosed herein, in certain embodiments, are pharmaceuticalcompositions comprising a selective delivery molecule according toSDM-64. Disclosed herein, in certain embodiments, are pharmaceuticalcompositions comprising a selective delivery molecule according toSDM-65.

Disclosed herein, in certain embodiments, are pharmaceuticalcompositions comprising a selective delivery molecule of Formula I,having the structure:

[c _(M)-M]-[[D_(A)-c _(A)]-(A-X-B)-[c _(B)-D_(B)]]  Formula I

wherein,

-   -   X is a cleavable linker;    -   A is a peptide with a sequence comprising 5 to 9 acidic amino        acids;    -   B is a peptide with a sequence comprising 7 to 9 basic amino        acids;    -   c_(A), c_(B), and c_(M) are independently 0-1 amino acid;    -   M is a macromolecule carrier; and    -   D_(A) and D_(B) are each independently selected from an imaging        agent and a therapeutic; and        wherein [c_(M)-M] is bound at any position on or between A, X,        and B, [D_(A)-c_(A)] is bound to any amino acid on A or X, and        [c_(B)-D_(B)] is bound to any amino acid on B.        In some embodiments, A and B do not have an equal number of        acidic and basic amino acids. In some embodiments, the number of        basic amino acids in B is greater than the number of acidic        amino acids in A. In some embodiments, A is a peptide comprising        5 or 9 consecutive glutamates (SEQ ID NOS: 4-5, respectively).        In some embodiments, B is a peptide comprising 8 or 9        consecutive arginines (SEQ ID NOS: 6-7, respectively). In some        embodiments, A is a peptide comprising 5 or 9 consecutive        glutamates (SEQ ID NOS: 4-5, respectively) and B is a peptide        comprising 8 or 9 consecutive arginines (SEQ ID NOS: 6-7,        respectively). In some embodiments, A is a peptide comprising 5        consecutive glutamates (SEQ ID NO: 4) and B is a peptide        comprising 8 consecutive arginines (SEQ ID NO: 6). In some        embodiments, c_(A), c_(B), and c_(M) are each independently a        0-1 amino acid. In some embodiments, c_(A), c_(B), and c_(M) are        each independently selected from a naturally-occurring amino        acid or a non-naturally-occurring amino acid. In some        embodiments, c_(A), c_(B), and c_(M) are each independently        selected from a D amino acid, a L amino acid, an α-amino acid, a        β-amino acid, or a δ-amino acid. In some embodiments, c_(A),        c_(B), and c_(M) are each independently selected from any amino        acid having a free thiol group, any amino acid having a        N-terminal amine group, and any amino acid with a side chain        capable of forming an oxime or hydrazone bond upon reaction with        a hydroxylamine or hydrazine group. In some embodiments, c_(A),        c_(B), and c_(M) are each independently selected from        D-cysteine, D-glutamate, lysine, and para-4-acetyl        L-phenylalanine. In some embodiments, c_(B) is any amino acid        having a free thiol group. In some embodiments, c_(B) is        D-cysteine. In some embodiments, c_(A) is any amino acid having        a N-terminal amine group. In some embodiments, c_(A) is        D-glutamate. In some embodiments, c_(A) is lysine. In some        embodiments, c_(M) is any amino acid with a side chain capable        of forming an oxime or hydrazone bond upon reaction with a        hydroxylamine or hydrazine group. In some embodiments, c_(M) is        para-4-acetyl L-phenylalanine. In some embodiments, X is        cleavable by a protease. In some embodiments, X is cleavable by        a matrix metalloproteinase. In some embodiments, X comprises an        amino acid sequence that is cleavable by MMP2, MMP7, MMP9, or        MMP14. In some embodiments, X comprises a peptide linkage. In        some embodiments, X comprises an amino acid sequence selected        from: PLGLAG (SEQ ID NO: 2), PLG-C(me)-AG (SEQ ID NO: 1),        RPLALWRS (SEQ ID NO: 3), ESPAYYTA (SEQ ID NO: 8), DPRSFL (SEQ ID        NO: 9), PPRSFL (SEQ ID NO: 10), RLQLKL (SEQ ID NO: 11), and        RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, X comprises the        amino acid sequence PLGLAG (SEQ ID NO: 2). In some embodiments,        X comprises the amino acid sequence PLG-C(me)-AG (SEQ ID NO: 1).        In some embodiments, X comprises the amino acid sequence        RPLALWRS (SEQ ID NO: 3). In some embodiments, X comprises the        amino acid sequence DPRSFL (SEQ ID NO: 9). In some embodiments,        X comprises the amino acid sequence RLQLKL (SEQ ID NO: 11). In        some embodiments, X comprises the amino acid sequence RLQLK(Ac)        (SEQ ID NO: 12). In some embodiments, M is selected from a        protein, a natural polymer, a synthetic polymer, or a dendrimer.        In some embodiments, M is selected from dextran, PEG polymers,        albumin, or a combination thereof. In some embodiments, M is PEG        polymers. In some embodiments, M is selected from PEG polymers        having an average molecular weight of approximately 0.5 kDa (PEG        0.5 kDa), PEG polymers having an average molecular weight of        approximately 2 kDa (PEG 2 kDa), PEG polymers having an average        molecular weight of approximately 5 kDa (PEG 5 kDa), PEG        polymers having an average molecular weight of approximately 12        kDa (PEG 12 kDa), PEG polymers having an average molecular        weight of approximately 20 kDa (PEG 20 kDa), PEG polymers having        an average molecular weight of approximately 30 kDa (PEG 30        kDa), and PEG polymers having an average molecular weight of        approximately 40 kDa (PEG40 kDa). In some embodiments, D_(A) and        D_(B) are a pair of acceptor and donor fluorescent moieties that        are capable of undergoing Försters/fluorescence resonance energy        transfer with the other. In some embodiments, D_(A) and D_(B)        are indocarbocyanine dyes. In some embodiments, D_(A) and D_(B)        are Cy5 and Cy7. In some embodiments, D_(A) and D_(B) are Cy5        and IRDye750. In some embodiments, D_(A) and D_(B) are Cy5 and        IRDye800. In some embodiments, D_(A) and D_(B) are Cy5 and ICG.        In some embodiments, D_(A) and D_(B) are a fluorescent moiety        and a fluorescence-quenching moiety. In some embodiments, the        molecule of Formula I is: SDM-41, SDM-42, SDM-43, SDM-44,        SDM-45, SDM-46, SDM-47, SDM-48, SDM-49, SDM-50, SDM-51, SDM-52,        SDM-53, SDM-54, SDM-55, SDM-56, SDM-57, SDM-58, SDM-59, SDM-60,        SDM-61, SDM-62, SDM-63, SDM-64, or SDM-65. In some embodiments,        the molecule is SDM-41. In some embodiments, the molecule of        Formula I is SDM-42.

Disclosed herein, in certain embodiments, are pharmaceuticalcompositions comprising a selective delivery molecule of Formula I,having the structure:

[c _(M)-M]-[[D_(A)-c _(A)]-(A-X-B)-[c _(B)-D_(B)]]  Formula I

wherein,

-   -   X is a cleavable linker;    -   A is a peptide with a sequence comprising 5 to 9 acidic amino        acids;    -   B is a peptide with a sequence comprising 7 to 9 basic amino        acids;    -   c_(A), c_(B), and c_(M) are independently 0-1 amino acid;    -   M is a polyethylene glycol (PEG) polymer; and    -   D_(A) and D_(B) are each independently an imaging agent; and    -   wherein [c_(M)-M] is bound to at any position on A or X,        [D_(A)-c_(A)] is bound to any amino acid on A or X, and        [c_(B)-D_(B)] is bound to any amino acid on B.        In some embodiments, A and B do not have an equal number of        acidic and basic amino acids. In some embodiments, the number of        basic amino acids in B is greater than the number of acidic        amino acids in A. In some embodiments, A is a peptide comprising        5 or 9 consecutive glutamates (SEQ ID NOS: 4-5, respectively).        In some embodiments, B is a peptide comprising 8 or 9        consecutive arginines (SEQ ID NO: 6-7, respectively). In some        embodiments, A is a peptide comprising 5 or 9 consecutive        glutamates (SEQ ID NOS: 4-5, respectively) and B is a peptide        comprising 8 or 9 consecutive arginines (SEQ ID NO: 6-7,        respectively). In some embodiments, A is a peptide comprising 5        consecutive glutamates (SEQ ID NO: 4) and B is a peptide        comprising 8 consecutive arginines (SEQ ID NO: 6). In some        embodiments, c_(A), c_(B), and c_(M) are each independently a        0-1 amino acid. In some embodiments, c_(A), c_(B), and c_(M) are        each independently selected from a naturally-occurring amino        acid or a non-naturally-occurring amino acid. In some        embodiments, c_(A), c_(B), and c_(M) are each independently        selected from a D amino acid, a L amino acid, an α-amino acid, a        β-amino acid, or a δ-amino acid. In some embodiments, c_(A),        c_(B), and c_(M) are each independently selected from any amino        acid having a free thiol group, any amino acid having a        N-terminal amine group, and any amino acid with a side chain        capable of forming an oxime or hydrazone bond upon reaction with        a hydroxylamine or hydrazine group. In some embodiments, c_(A),        c_(B), and c_(M) are each independently selected from        D-cysteine, D-glutamate, lysine, and para-4-acetyl        L-phenylalanine. In some embodiments, c_(B) is any amino acid        having a free thiol group. In some embodiments, c_(B) is        D-cysteine. In some embodiments, c_(A) is any amino acid having        a N-terminal amine group. In some embodiments, c_(A) is        D-glutamate. In some embodiments, c_(A) is lysine. In some        embodiments, c_(M) is any amino acid with a side chain capable        of forming an oxime or hydrazone bond upon reaction with a        hydroxylamine or hydrazine group. In some embodiments, c_(M) is        para-4-acetyl L-phenylalanine. In some embodiments, X is        cleavable by a protease. In some embodiments, X is cleavable by        a matrix metalloproteinase. In some embodiments, X comprises an        amino acid sequence that is cleavable by MMP2, MMP7, MMP9, or        MMP14. In some embodiments, X comprises a peptide linkage. In        some embodiments, X comprises an amino acid sequence selected        from: PLGLAG (SEQ ID NO: 2), PLG-C(me)-AG (SEQ ID NO: 1),        RPLALWRS (SEQ ID NO: 3), ESPAYYTA (SEQ ID NO: 8), DPRSFL (SEQ ID        NO: 9), PPRSFL (SEQ ID NO: 10), RLQLKL (SEQ ID NO: 11), and        RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, X comprises the        amino acid sequence PLGLAG (SEQ ID NO: 2). In some embodiments,        X comprises the amino acid sequence PLG-C(me)-AG (SEQ ID NO: 1).        In some embodiments, X comprises the amino acid sequence        RPLALWRS (SEQ ID NO: 3). In some embodiments, X comprises the        amino acid sequence DPRSFL (SEQ ID NO: 9). In some embodiments,        X comprises the amino acid sequence PPRSFL (SEQ ID NO: 10). In        some embodiments, X comprises the amino acid sequence RLQLKL        (SEQ ID NO: 11). In some embodiments, X comprises the amino acid        sequence RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, D_(A)        and D_(B) are a pair of acceptor and donor fluorescent moieties        that are capable of undergoing Försters/fluorescence resonance        energy transfer with the other. In some embodiments, D_(A) and        D_(B) are Cy5 and Cy7. In some embodiments, D_(A) and D_(B) are        Cy5 and IRDye750. In some embodiments, D_(A) and D_(B) are Cy5        and IRDye800. In some embodiments, D_(A) and D_(B) are Cy5 and        ICG. In some embodiments, D_(A) and D_(B) are a fluorescent        moiety and a fluorescence-quenching moiety. In some embodiments,        the molecule of Formula I is: SDM-41, SDM-42, SDM-43, SDM-44,        SDM-45, SDM-46, SDM-47, SDM-48, SDM-49, SDM-50, SDM-51, SDM-52,        SDM-53, SDM-54, SDM-55, SDM-56, SDM-57, SDM-58, SDM-59, SDM-60,        SDM-61, SDM-62, SDM-63, SDM-64, or SDM-65. In some embodiments,        the molecule is SDM-41. In some embodiments, the molecule of        Formula I is SDM-42.

Pharmaceutical compositions herein are formulated using one or morephysiologically acceptable carriers including excipients and auxiliarieswhich facilitate processing of the active agents into preparations whichare used pharmaceutically. Proper formulation is dependent upon theroute of administration chosen. A summary of pharmaceutical compositionsis found, for example, in Remington: The Science and Practice ofPharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995);Hoover, John E., Remington's Pharmaceutical Sciences, Mack PublishingCo., Easton, Pa. 1975; Liberman, H. A. and Lachman, L., Eds.,Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; andPharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed.(Lippincott Williams & Wilkins, 1999).

In certain embodiments, a pharmaceutical composition disclosed hereinfurther comprises a pharmaceutically acceptable diluent(s),excipient(s), or carrier(s). In some embodiments, the pharmaceuticalcompositions includes other medicinal or pharmaceutical agents,carriers, adjuvants, such as preserving, stabilizing, wetting oremulsifying agents, solution promoters, salts for regulating the osmoticpressure, and/or buffers. In addition, the pharmaceutical compositionsalso contain other therapeutically valuable substances.

In certain embodiments, a pharmaceutical composition disclosed herein isadministered to a subject by any suitable administration route,including but not limited to, parenteral (intravenous, subcutaneous,intraperitoneal, intramuscular, intravascular, intrathecal,intravitreal, infusion, or local) administration.

Formulations suitable for intramuscular, subcutaneous, peritumoral, orintravenous injection include physiologically acceptable sterile aqueousor non-aqueous solutions, dispersions, suspensions or emulsions, andsterile powders for reconstitution into sterile injectable solutions ordispersions. Examples of suitable aqueous and non-aqueous carriers,diluents, solvents, or vehicles including water, ethanol, polyols(propyleneglycol, polyethylene-glycol, glycerol, cremophor and thelike), suitable mixtures thereof, vegetable oils (such as olive oil) andinjectable organic esters such as ethyl oleate. Proper fluidity ismaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case ofdispersions, and by the use of surfactants. Formulations suitable forsubcutaneous injection also contain optional additives such aspreserving, wetting, emulsifying, and dispensing agents.

For intravenous injections, an active agent is optionally formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hank's solution, Ringer's solution, or physiological saline buffer.

Parenteral injections optionally involve bolus injection or continuousinfusion. Formulations for injection are optionally presented in unitdosage form, e.g., in ampoules or in multi dose containers, with anadded preservative. In some embodiments, the pharmaceutical compositiondescribed herein are in a form suitable for parenteral injection as asterile suspensions, solutions or emulsions in oily or aqueous vehicles,and contain formulatory agents such as suspending, stabilizing and/ordispersing agents. Pharmaceutical formulations for parenteraladministration include aqueous solutions of an active agent in watersoluble form. Additionally, suspensions are optionally prepared asappropriate oily injection suspensions.

In some embodiments, the pharmaceutical composition described herein isin unit dosage forms suitable for single administration of precisedosages. In unit dosage form, the formulation is divided into unit dosescontaining appropriate quantities of an active agent disclosed herein.In some embodiments, the unit dosage is in the form of a packagecontaining discrete quantities of the formulation. Non-limiting examplesare packaged tablets or capsules, and powders in vials or ampoules. Insome embodiments, aqueous suspension compositions are packaged insingle-dose non-reclosable containers. Alternatively, multiple-dosereclosable containers are used, in which case it is typical to include apreservative in the composition. By way of example only, formulationsfor parenteral injection are presented in unit dosage form, whichinclude, but are not limited to ampoules, or in multi dose containers,with an added preservative.

Methods of Use

Selective delivery molecules SDM-41, SDM-42, SDM-43, SDM-44, SDM-45,SDM-46, SDM-47, SDM-48, SDM-49, SDM-50, SDM-51, SDM-52, SDM-53, SDM-54,SDM-55, SDM-56, SDM-57, SDM-58, SDM-59, SDM-60, SDM-61, SDM-62, SDM-63,SDM-64, or SDM-65 allow the targeted delivery of cargo to specific cellsand/or tissues.

Disclosed herein, in certain embodiments, are methods of delivering acargo to a tissue of interest, comprising contacting the tissue ofinterest with a molecule selected from SDM-41, SDM-42, SDM-43, SDM-44,SDM-45, SDM-46, SDM-47, SDM-48, SDM-49, SDM-50, SDM-51, SDM-52, SDM-53,SDM-54, SDM-55, SDM-56, SDM-57, SDM-58, SDM-59, SDM-60, SDM-61, SDM-62,SDM-63, SDM-64, or SDM-65. In some embodiments, the molecule is SDM-41.

Disclosed herein, in certain embodiments, are methods of delivering acargo to a tissue of interest, comprising contacting the tissue ofinterest with a molecule of Formula I:

[c _(M)-M]-[[D_(A)-c _(A)]-(A-X-B)-[c _(B)-D_(B)]]  Formula I

wherein,

-   -   X is a cleavable linker;    -   A is a peptide with a sequence comprising 5 to 9 acidic amino        acids;    -   B is a peptide with a sequence comprising 7 to 9 basic amino        acids;    -   c_(A), c_(B), and c_(M) are independently 0-1 amino acid;    -   M is a macromolecule carrier; and    -   D_(A) and D_(B) are each independently selected from an imaging        agent and a therapeutic; and        wherein [c_(M)-M] is bound at any position on or between A, X,        and B, [D_(A)-c_(A)] is bound to any amino acid on A or X, and        [c_(B)-D_(B)] is bound to any amino acid on B. In some        embodiments, A and B do not have an equal number of acidic and        basic amino acids. In some embodiments, the number of basic        amino acids in B is greater than the number of acidic amino        acids in A. In some embodiments, A is a peptide comprising 5 or        9 consecutive glutamates (SEQ ID NOS: 4-5, respectively). In        some embodiments, B is a peptide comprising 8 or 9 consecutive        arginines (SEQ ID NOS: 6-7, respectively). In some embodiments,        A is a peptide comprising 5 or 9 consecutive glutamates (SEQ ID        NOS: 4-5, respectively) and B is a peptide comprising 8 or 9        consecutive arginines (SEQ ID NOS: 6-7, respectively). In some        embodiments, A is a peptide comprising 5 consecutive glutamates        (SEQ ID NO: 4) and B is a peptide comprising 8 consecutive        arginines (SEQ ID NO: 6). In some embodiments, c_(A), c_(B), and        c_(M) are each independently a 0-1 amino acid. In some        embodiments, c_(A), c_(B), and c_(M) are each independently        selected from a naturally-occurring amino acid or a        non-naturally-occurring amino acid. In some embodiments, c_(A),        c_(B), and c_(M) are each independently selected from a D amino        acid, a L amino acid, an α-amino acid, a β-amino acid, or a        δ-amino acid. In some embodiments, c_(A), c_(B), and c_(M) are        each independently selected from any amino acid having a free        thiol group, any amino acid having a N-terminal amine group, and        any amino acid with a side chain capable of forming an oxime or        hydrazone bond upon reaction with a hydroxylamine or hydrazine        group. In some embodiments, c_(A), c_(B), and c_(M) are each        independently selected from D-cysteine, D-glutamate, lysine, and        para-4-acetyl L-phenylalanine. In some embodiments, c_(B) is any        amino acid having a free thiol group. In some embodiments, c_(B)        is D-cysteine. In some embodiments, c_(A) is any amino acid        having a N-terminal amine group. In some embodiments, c_(A) is        D-glutamate. In some embodiments, c_(A) is lysine. In some        embodiments, c_(M) is any amino acid with a side chain capable        of forming an oxime or hydrazone bond upon reaction with a        hydroxylamine or hydrazine group. In some embodiments, c_(M) is        para-4-acetyl L-phenylalanine. In some embodiments, X is        cleavable by a protease. In some embodiments, X is cleavable by        a matrix metalloproteinase. In some embodiments, X comprises an        amino acid sequence that is cleavable by MMP2, MMP7, MMP9, or        MMP14. In some embodiments, X comprises a peptide linkage. In        some embodiments, X comprises an amino acid sequence selected        from: PLGLAG (SEQ ID NO: 2), PLG-C(me)-AG (SEQ ID NO:1),        RPLALWRS (SEQ ID NO: 3), ESPAYYTA (SEQ ID NO: 8), DPRSFL (SEQ ID        NO: 9), PPRSFL (SEQ ID NO: 10), RLQLKL (SEQ ID NO: 11), and        RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, X comprises the        amino acid sequence PLGLAG (SEQ ID NO: 2). In some embodiments,        X comprises the amino acid sequence PLG-C(me)-AG (SEQ ID NO: 1).        In some embodiments, X comprises the amino acid sequence        RPLALWRS (SEQ ID NO: 3). In some embodiments, X comprises the        amino acid sequence DPRSFL (SEQ ID NO: 9). In some embodiments,        X comprises the amino acid sequence RLQLKL (SEQ ID NO: 11). In        some embodiments, X comprises the amino acid sequence RLQLK(Ac)        (SEQ ID NO: 12). In some embodiments, M is selected from a        protein, a natural polymer, a synthetic polymer, or a dendrimer.        In some embodiments, M is selected from dextran, PEG polymers,        albumin, or a combination thereof. In some embodiments, M is PEG        polymers. In some embodiments, M is selected from PEG polymers        having an average molecular weight of approximately 0.5 kDa (PEG        0.5 kDa), PEG polymers having an average molecular weight of        approximately 2 kDa (PEG 2 kDa), PEG polymers having an average        molecular weight of approximately 5 kDa (PEG 5 kDa), PEG        polymers having an average molecular weight of approximately 12        kDa (PEG 12 kDa), PEG polymers having an average molecular        weight of approximately 20 kDa (PEG 20 kDa), PEG polymers having        an average molecular weight of approximately 30 kDa (PEG 30        kDa), and PEG polymers having an average molecular weight of        approximately 40 kDa (PEG40 kDa). In some embodiments, D_(A) and        D_(B) are a pair of donor and acceptor fluorescent moieties that        are capable of undergoing Försters/fluorescence resonance energy        transfer with the other. In some embodiments, D_(A) and D_(B)        are indocarbocyanine dyes. In some embodiments, D_(A) and D_(B)        are Cy5 and Cy7. In some embodiments, D_(A) and D_(B) are Cy5        and IRDye750. In some embodiments, D_(A) and D_(B) are Cy5 and        IRDye800. In some embodiments, D_(A) and D_(B) are Cy5 and ICG.        In some embodiments, D_(A) and D_(B) are a fluorescent moiety        and a fluorescence-quenching moiety. In some embodiments, the        molecule of Formula I is: SDM-41, SDM-42, SDM-43, SDM-44,        SDM-45, SDM-46, SDM-47, SDM-48, SDM-49, SDM-50, SDM-51, SDM-52,        SDM-53, SDM-54, SDM-55, SDM-56, SDM-57, SDM-58, SDM-59, SDM-60,        SDM-61, SDM-62, SDM-63, SDM-64, or SDM-65. In some embodiments,        the molecule is SDM-41. In some embodiments, the molecule of        Formula I is SDM-42.

Tissue of Interest

In some embodiments, the tissue of interest is cancerous tissue (or,cancer). In some embodiments, the cancerous tissue is: breast cancertissue, colorectal cancer tissue, squamous cell carcinoma tissue, skincancer tissue, prostate cancer tissue, melanoma tissue, ovarian cancertissue, cancerous lymph node tissue, or thyroid cancer tissue. In someembodiments, the cancerous tissue is breast cancer tissue. In someembodiments, the cancerous tissue is colorectal cancer tissue. In someembodiments, the cancerous tissue is cancerous lymph node tissue. Insome embodiments, the cancerous tissue is squamous cell carcinomatissue. In some embodiments, the cancerous tissue is skin cancer tissue.

In some embodiments, the cancer is AIDS-related cancers (e.g.,AIDS-related lymphoma), anal cancer, basal cell carcinoma, bile ductcancer (e.g., extrahepatic), bladder cancer, bone cancer, (osteosarcomaand malignant fibrous histiocytoma), breast cancer, cervical cancer,colorectal cancer, endometrial cancer (e.g., uterine cancer),ependymoma, esophageal cancer, eye cancer (e.g., intraocular melanomaand retinoblastoma), gastric (stomach) cancer, germ cell tumor, (e.g.,extracranial, extragonadal, ovarian), head and neck cancer, leukemia,lip and oral cavity cancer, liver cancer, lung cancer (e.g., small celllung cancer, non-small cell lung cancer, adenocarcinoma of the lung, andsquamous carcinoma of the lung), ovarian cancer, pancreatic cancer,pituitary tumor, prostate cancer, renal cancer, skin cancer, smallintestine cancer, squamous cell cancer, testicular cancer, throatcancer, thyroid cancer, urethral cancer, and post-transplantlymphoproliferative disorder (PTLD).

In some embodiments, the cancer is a lymphoid cancer (e.g., lymphoma).

In some embodiments, the cancer is a B-cell cancer. In some embodiments,the cancer is precursor B-cell cancers (e.g., precursor B-lymphoblasticleukemia/lymphoma) and peripheral B-cell cancers (e.g., B-cell chroniclymphocytic leukemia/prolymphocytic leukemia/small lymphocytic lymphoma(small lymphocytic (SL) NHL), lymphoplasmacytoid lymphoma/immunocytoma,mantel cell lymphoma, follicle center lymphoma, follicular lymphoma(e.g., cytologic grades: I (small cell), II (mixed small and largecell), III (large cell) and/or subtype: diffuse and predominantly smallcell type), low grade/follicular non-Hodgkin's lymphoma (NHL),intermediate grade/follicular NHL, marginal zone B-cell lymphoma (e.g.,extranodal (e.g., MALT-type+/−monocytoid B cells) and/or Nodal (e.g.,+/−monocytoid B cells)), splenic marginal zone lymphoma (e.g.,+/−villous lymphocytes), Hairy cell leukemia, plasmacytoma/plasma cellmyeloma (e.g., myeloma and multiple myeloma), diffuse large B-celllymphoma (e.g., primary mediastinal (thymic) B-cell lymphoma),intermediate grade diffuse NHL, Burkitt's lymphoma, High-grade B-celllymphoma, Burkitt-like, high grade immunoblastic NHL, high gradelymphoblastic NHL, high grade small non-cleaved cell NHL, bulky diseaseNHL, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia).

In some embodiments, the cancer is a T-cell and/or putative NK-cellcancer. In some embodiments, the cancer is precursor T-cell cancer(precursor T-lymphoblastic lymphoma/leukemia) and peripheral T-cell andNK-cell cancers (e.g., T-cell chronic lymphocyticleukemia/prolymphocytic leukemia, and large granular lymphocyte leukemia(LGL) (e.g., T-cell type and/or NK-cell type), cutaneous T-cell lymphoma(e.g., mycosis fungoides/Sezary syndrome), primary T-cell lymphomasunspecified (e.g., cytological categories (e.g., medium-sized cell,mixed medium and large cell), large cell, lymphoepitheloid cell, subtypehepatosplenic γδ T-cell lymphoma, and subcutaneous panniculitic T-celllymphoma), angioimmunoblastic T-cell lymphoma (AILD), angiocentriclymphoma, intestinal T-cell lymphoma (e.g., +/−enteropathy associated),adult T-cell lymphoma/leukemia (ATL), anaplastic large cell lymphoma(ALCL) (e.g., CD30+, T- and null-cell types), anaplastic large-celllymphoma, and Hodgkin's like).

In some embodiments, the cancer is Hodgkin's disease.

In some embodiments, the cancer is leukemia. In some embodiments, thecancer is chronic myelocytic I (granulocytic) leukemia, chronicmyelogenous, and chronic lymphocytic leukemia (CLL), acute lymphoblasticleukemia (ALL), acute myeloid leukemia, acute lymphocytic leukemia, andacute myelocytic leukemia (e.g., myeloblastic, promyelocytic,myelomonocytic, monocytic, and erythroleukemia).

In some embodiments, the cancer is a liquid tumor or plasmacytoma. Insome embodiments, the cancer is extramedullary plasmacytoma, a solitarymyeloma, and multiple myeloma. In some embodiments, the plasmacytoma ismultiple myeloma.

In some embodiments, the cancer is lung cancer.

In some embodiments, the cancer is prostate cancer. In some embodiments,the prostate cancer is an adenocarcinoma. In some embodiments, theprostate cancer is a sarcoma, neuroendocrine tumor, small cell cancer,ductal cancer, or a lymphoma. In some embodiments, the prostate canceris stage A prostate cancer (the cancer cannot be felt during a rectalexam). In some embodiments, the prostate cancer is stage B prostatecancer (i.e., the tumor involves more tissue within the prostate, it canbe felt during a rectal exam, or it is found with a biopsy that is donebecause of a high PSA level). In some embodiments, the prostate canceris stage C prostate cancer (i.e., the cancer has spread outside theprostate to nearby tissues). In some embodiments, the prostate cancer isstage D prostate cancer. In some embodiments, the prostate cancer isandrogen independent prostate cancer (AIPC). In some embodiments, theprostate cancer is androgen dependent prostate cancer. In someembodiments, the prostate cancer is refractory to hormone therapy. Insome embodiments, the prostate cancer is substantially refractory tohormone therapy. In some embodiments, the prostate cancer is refractoryto chemotherapy. In some embodiments, the prostate cancer is metastaticprostate cancer. In some embodiments, the individual is a human who hasa gene, genetic mutation, or polymorphism associated with prostatecancer (e.g., RNASEL/HPC1, ELAC2/HPC2, SR-A/MSR1, CHEK2, BRCA2, PON1,OGG1, MIC-1, TLR4, and PTEN) or has one or more extra copies of a geneassociated with prostate cancer. In some embodiments, the prostatecancer is HER2 positive. In some embodiments, the prostate cancer isHER2 negative.

In some embodiments, the cancer has metastasized and is characterized bycirculating tumor cells.

Imaging Uses

The selective delivery molecules SDM-41, SDM-42, SDM-43, SDM-44, SDM-45,SDM-46, SDM-47, SDM-48, SDM-49, SDM-50, SDM-51, SDM-52, SDM-53, SDM-54,SDM-55, SDM-56, SDM-57, SDM-58, SDM-59, SDM-60, SDM-61, SDM-62, SDM-63,SDM-64, and SDM-65 allow the targeted delivery of imaging agents tospecific cells and/or tissues (e.g., cancerous tissues). In someembodiments, the selective delivery molecules enable targeted deliveryof one or more imaging agents to a cell or tissue. In some embodiments,targeted delivery of an imaging agent to a cell or tissue enables amedical professional to visualize/image a specific tissue.

Disclosed herein, in certain embodiments, are methods of deliveringimaging agents to a tissue of interest, comprising contacting the tissueof interest with a molecule selected from the group consisting of:SDM-41, SDM-42, SDM-43, SDM-44, SDM-45, SDM-46, SDM-47, SDM-48, SDM-49,SDM-50, SDM-51, SDM-52, SDM-53, SDM-54, SDM-55, SDM-56, SDM-57, SDM-58,SDM-59, SDM-60, SDM-61, SDM-62, SDM-63, SDM-64, and SDM-65. In someembodiments, the molecule is SDM-41.

Disclosed herein, in certain embodiments, are methods of deliveringimaging agents to a tissue of interest, comprising contacting the tissueof interest with a molecule of Formula I:

[c _(M)-M]-[[D_(A)-c _(A)]-(A-X-B)-[c _(B)-D_(B)]]  Formula I

wherein,

-   -   X is a peptide linker cleavable by a matrix metalloproteinase;    -   A is a peptide with a sequence comprising 5 or 9 consecutive        glutamates (SEQ ID NOS: 4-5, respectively);    -   B is a peptide with a sequence comprising 8 or 9 consecutive        arginines (SEQ ID NOS: 6-7, respectively);    -   c_(A), c_(B), and c_(M) are independently 0-1 amino acid;    -   M is a polyethylene glycol (PEG) polymer; and    -   D_(A) and D_(B) are each independently an imaging agent; and        wherein [c_(M)-M] is bound to at any position on A or X,        [D_(A)-c_(A)] is bound to any amino acid on A or X, and        [c_(B)-D_(B)] is bound to any amino acid on B.        In some embodiments, A and B do not have an equal number of        acidic and basic amino acids. In some embodiments, c_(A), c_(B),        and c_(M) are each independently a 0-1 amino acid. In some        embodiments, c_(A), c_(B), and c_(M) are each independently        selected from any amino acid having a free thiol group, any        amino acid having a N-terminal amine group, and any amino acid        with a side chain capable of forming an oxime or hydrazone bond        upon reaction with a hydroxylamine or hydrazine group. In some        embodiments, c_(A), c_(B), and c_(M) are each independently        selected from D-cysteine, D-glutamate, lysine, and para-4-acetyl        L-phenylalanine. In some embodiments, c_(B) is any amino acid        having a free thiol group. In some embodiments, c_(B) is        D-cysteine. In some embodiments, c_(A) is any amino acid having        a N-terminal amine group. In some embodiments, c_(A) is        D-glutamate. In some embodiments, c_(M) is any amino acid with a        side chain capable of forming an oxime or hydrazone bond upon        reaction with a hydroxylamine or hydrazine group. In some        embodiments, c_(M) is para-4-acetyl L-phenylalanine. In some        embodiments, X comprises the amino acid sequence RPLALWRS (SEQ        ID NO: 3). In some embodiments, X comprises the amino acid        sequence DPRSFL (SEQ ID NO: 9). In some embodiments, X comprises        the amino acid sequence PPRSFL (SEQ ID NO: 10). In some        embodiments, X comprises the amino acid sequence RLQLKL (SEQ ID        NO: 11). In some embodiments, X comprises the amino acid        sequence RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, D_(A)        and D_(B) are a pair of acceptor and donor fluorescent moieties        that are capable of undergoing Försters/fluorescence resonance        energy transfer with the other. In some embodiments, D_(A) and        D_(B) are indocarbocyanine dyes. In some embodiments, D_(A) and        D_(B) are Cy5 and Cy7. In some embodiments, D_(A) and D_(B) are        Cy5 and IRDye750. In some embodiments, D_(A) and D_(B) are Cy5        and IRDye800. In some embodiments, D_(A) and D_(B) are Cy5 and        ICG. In some embodiments, D_(A) and D_(B) are a fluorescent        moiety and a fluorescence-quenching moiety.

Disclosed herein, in certain embodiments, are methods of delivering apair of acceptor and donor fluorescent moieties that are capable ofundergoing Försters/fluorescence resonance energy transfer with theother to a tissue of interest, comprising contacting the tissue ofinterest with a molecule of Formula I:

[c _(M)-M]-[[D_(A)-c _(A)]-(A-X-B)-[c _(B)-D_(B)]]  Formula I

wherein,

-   -   X is a cleavable linker;    -   A is a peptide with a sequence comprising 5 to 9 acidic amino        acids;    -   B is a peptide with a sequence comprising 7 to 9 basic amino        acids;    -   c_(A), c_(B), and c_(M) are independently 0-1 amino acid;    -   M is a polyethylene glycol (PEG) polymer; and    -   D_(A) and D_(B) are a pair of acceptor and donor fluorescent        moieties that are capable of undergoing Försters/fluorescence        resonance energy transfer with the other; and    -   wherein [c_(M)-M] is bound to at any position on A or X,        [D_(A)-c_(A)] is bound to any amino acid on A or X, and        [c_(B)-D_(B)] is bound to any amino acid on B.        In some embodiments, A and B do not have an equal number of        acidic and basic amino acids. In some embodiments, the number of        basic amino acids in B is greater than the number of acidic        amino acids in A. In some embodiments, A is a peptide comprising        5 or 9 consecutive glutamates (SEQ ID NOS: 4-5, respectively).        In some embodiments, B is a peptide comprising 8 or 9        consecutive arginines (SEQ ID NOS: 6-7, respectively). In some        embodiments, A is a peptide comprising 5 or 9 consecutive        glutamates (SEQ ID NOS: 4-5, respectively) and B is a peptide        comprising 8 or 9 consecutive arginines (SEQ ID NOS: 6-7,        respectively). In some embodiments, A is a peptide comprising 5        consecutive glutamates (SEQ ID NO: 4) and B is a peptide        comprising 8 consecutive arginines (SEQ ID NO: 6). In some        embodiments, c_(A), c_(B), and c_(M) are each independently a        0-1 amino acid. In some embodiments, c_(A), c_(B), and c_(M) are        each independently selected from a naturally-occurring amino        acid or a non-naturally-occurring amino acid. In some        embodiments, c_(A), c_(B), and c_(M) are each independently        selected from a D amino acid, a L amino acid, an α-amino acid, a        β-amino acid, or a δ-amino acid. In some embodiments, c_(A),        c_(B), and c_(M) are each independently selected from any amino        acid having a free thiol group, any amino acid having a        N-terminal amine group, and any amino acid with a side chain        capable of forming an oxime or hydrazone bond upon reaction with        a hydroxylamine or hydrazine group. In some embodiments, c_(A),        c_(B), and c_(M) are each independently selected from        D-cysteine, D-glutamate, lysine, and para-4-acetyl        L-phenylalanine. In some embodiments, c_(B) is any amino acid        having a free thiol group. In some embodiments, c_(B) is        D-cysteine. In some embodiments, c_(A) is any amino acid having        a N-terminal amine group. In some embodiments, c_(A) is        D-glutamate. In some embodiments, c_(A) is lysine. In some        embodiments, c_(M) is any amino acid with a side chain capable        of forming an oxime or hydrazone bond upon reaction with a        hydroxylamine or hydrazine group. In some embodiments, c_(M) is        para-4-acetyl L-phenylalanine. In some embodiments, X is        cleavable by a protease. In some embodiments, X is cleavable by        a matrix metalloproteinase. In some embodiments, X comprises an        amino acid sequence that is cleavable by MMP2, MMP7, MMP9, or        MMP14. In some embodiments, X comprises a peptide linkage. In        some embodiments, X comprises an amino acid sequence selected        from: PLGLAG (SEQ ID NO: 2), PLG-C(me)-AG (SEQ ID NO: 1),        RPLALWRS (SEQ ID NO: 3), ESPAYYTA (SEQ ID NO: 8), DPRSFL (SEQ ID        NO: 9), PPRSFL (SEQ ID NO: 10), RLQLKL (SEQ ID NO: 11), and        RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, X comprises the        amino acid sequence PLGLAG (SEQ ID NO: 2). In some embodiments,        X comprises the amino acid sequence PLG-C(me)-AG (SEQ ID NO: 1).        In some embodiments, X comprises the amino acid sequence        RPLALWRS (SEQ ID NO: 3). In some embodiments, X comprises the        amino acid sequence DPRSFL (SEQ ID NO: 9). In some embodiments,        X comprises the amino acid sequence PPRSFL (SEQ ID NO: 10). In        some embodiments, X comprises the amino acid sequence RLQLKL        (SEQ ID NO: 11). In some embodiments, X comprises the amino acid        sequence RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, D_(A)        and D_(B) are a pair of acceptor and donor fluorescent moieties        that are capable of undergoing Försters/fluorescence resonance        energy transfer with the other. In some embodiments, D_(A) and        D_(B) are Cy5 and Cy7. In some embodiments, D_(A) and D_(B) are        Cy5 and IRDye750. In some embodiments, D_(A) and D_(B) are Cy5        and IRDye800. In some embodiments, D_(A) and D_(B) are Cy5 and        ICG. In some embodiments, D_(A) and D_(B) are a fluorescent        moiety and a fluorescence-quenching moiety. In some embodiments,        the molecule of Formula I is: SDM-41, SDM-42, SDM-43, SDM-44,        SDM-45, SDM-46, SDM-47, SDM-48, SDM-49, SDM-50, SDM-51, SDM-52,        SDM-53, SDM-54, SDM-55, SDM-56, SDM-57, SDM-58, SDM-59, SDM-60,        SDM-61, SDM-62, SDM-63, SDM-64, or SDM-65. In some embodiments,        the molecule is SDM-41. In some embodiments, the molecule of        Formula I is SDM-42.

Disclosed herein, in certain embodiments, are methods of delivering apair of acceptor and donor fluorescent moieties that are capable ofundergoing Försters/fluorescence resonance energy transfer with theother to a tissue of interest, comprising contacting the tissue ofinterest with SDM-41.

Disclosed herein, in certain embodiments, are methods of delivering apair of acceptor and donor fluorescent moieties that are capable ofundergoing Försters/fluorescence resonance energy transfer with theother to a tissue of interest, comprising contacting the tissue ofinterest with SDM-42.

Disclosed herein, in certain embodiments, are methods of delivering apair of acceptor and donor fluorescent moieties that are capable ofundergoing Försters/fluorescence resonance energy transfer with theother to a tissue of interest, comprising contacting the tissue ofinterest with SDM-43.

Disclosed herein, in certain embodiments, are methods of delivering apair of acceptor and donor fluorescent moieties that are capable ofundergoing Försters/fluorescence resonance energy transfer with theother to a tissue of interest, comprising contacting the tissue ofinterest with SDM-44.

Disclosed herein, in certain embodiments, are methods of delivering apair of acceptor and donor fluorescent moieties that are capable ofundergoing Försters/fluorescence resonance energy transfer with theother to a tissue of interest, comprising contacting the tissue ofinterest with SDM-45.

Disclosed herein, in certain embodiments, are methods of delivering apair of acceptor and donor fluorescent moieties that are capable ofundergoing Försters/fluorescence resonance energy transfer with theother to a tissue of interest, comprising contacting the tissue ofinterest with SDM-46.

Disclosed herein, in certain embodiments, are methods of delivering apair of acceptor and donor fluorescent moieties that are capable ofundergoing Försters/fluorescence resonance energy transfer with theother o to a tissue of interest, comprising contacting the tissue ofinterest with SDM-47.

Disclosed herein, in certain embodiments, are methods of delivering apair of acceptor and donor fluorescent moieties that are capable ofundergoing Försters/fluorescence resonance energy transfer with theother to a tissue of interest, comprising contacting the tissue ofinterest with SDM-48.

Disclosed herein, in certain embodiments, are methods of delivering apair of acceptor and donor fluorescent moieties that are capable ofundergoing Försters/fluorescence resonance energy transfer with theother to a tissue of interest, comprising contacting the tissue ofinterest with SDM-49.

Disclosed herein, in certain embodiments, are methods of delivering apair of acceptor and donor fluorescent moieties that are capable ofundergoing Försters/fluorescence resonance energy transfer with theother to a tissue of interest, comprising contacting the tissue ofinterest with SDM-50.

Disclosed herein, in certain embodiments, are methods of delivering apair of acceptor and donor fluorescent moieties that are capable ofundergoing Försters/fluorescence resonance energy transfer with theother to a tissue of interest, comprising contacting the tissue ofinterest with SDM-51.

Disclosed herein, in certain embodiments, are methods of delivering apair of acceptor and donor fluorescent moieties that are capable ofundergoing Försters/fluorescence resonance energy transfer with theother to a tissue of interest, comprising contacting the tissue ofinterest with SDM-52.

Disclosed herein, in certain embodiments, are methods of delivering apair of acceptor and donor fluorescent moieties that are capable ofundergoing Försters/fluorescence resonance energy transfer with theother to a tissue of interest, comprising contacting the tissue ofinterest with SDM-53.

Disclosed herein, in certain embodiments, are methods of delivering apair of acceptor and donor fluorescent moieties that are capable ofundergoing Försters/fluorescence resonance energy transfer with theother to a tissue of interest, comprising contacting the tissue ofinterest with SDM-54.

Disclosed herein, in certain embodiments, are methods of delivering apair of acceptor and donor fluorescent moieties that are capable ofundergoing Försters/fluorescence resonance energy transfer with theother to a tissue of interest, comprising contacting the tissue ofinterest with SDM-55.

Disclosed herein, in certain embodiments, are methods of delivering apair of acceptor and donor fluorescent moieties that are capable ofundergoing Försters/fluorescence resonance energy transfer with theother to a tissue of interest, comprising contacting the tissue ofinterest with SDM-56.

Disclosed herein, in certain embodiments, are methods of delivering apair of acceptor and donor fluorescent moieties that are capable ofundergoing Försters/fluorescence resonance energy transfer with theother to a tissue of interest, comprising contacting the tissue ofinterest with SDM-57.

Disclosed herein, in certain embodiments, are methods of delivering apair of acceptor and donor fluorescent moieties that are capable ofundergoing Försters/fluorescence resonance energy transfer with theother to a tissue of interest, comprising contacting the tissue ofinterest with SDM-58.

Disclosed herein, in certain embodiments, are methods of delivering apair of acceptor and donor fluorescent moieties that are capable ofundergoing Försters/fluorescence resonance energy transfer with theother to a tissue of interest, comprising contacting the tissue ofinterest with SDM-59.

Disclosed herein, in certain embodiments, are methods of delivering apair of acceptor and donor fluorescent moieties that are capable ofundergoing Försters/fluorescence resonance energy transfer with theother to a tissue of interest, comprising contacting the tissue ofinterest with SDM-60.

Disclosed herein, in certain embodiments, are methods of delivering apair of acceptor and donor fluorescent moieties that are capable ofundergoing Försters/fluorescence resonance energy transfer with theother to a tissue of interest, comprising contacting the tissue ofinterest with SDM-61.

Disclosed herein, in certain embodiments, are methods of delivering apair of acceptor and donor fluorescent moieties that are capable ofundergoing Försters/fluorescence resonance energy transfer with theother to a tissue of interest, comprising contacting the tissue ofinterest with SDM-62.

Disclosed herein, in certain embodiments, are methods of delivering apair of acceptor and donor fluorescent moieties that are capable ofundergoing Försters/fluorescence resonance energy transfer with theother to a tissue of interest, comprising contacting the tissue ofinterest with SDM-63.

Disclosed herein, in certain embodiments, are methods of delivering apair of donor and acceptor fluorescent moieties that are capable ofundergoing Försters/fluorescence resonance energy transfer with theother to a tissue of interest, comprising contacting the tissue ofinterest with SDM-64.

Disclosed herein, in certain embodiments, are methods of delivering apair of acceptor and donor fluorescent moieties that are capable ofundergoing Försters/fluorescence resonance energy transfer with theother to a tissue of interest, comprising contacting the tissue ofinterest with SDM-65.

Disclosed herein, in certain embodiments, are methods of visualizing atissue of interest in an individual in need thereof, comprising: (a)administering to the individual a molecule selected from SDM-41, SDM-42,SDM-43, SDM-44, SDM-45, SDM-46, SDM-47, SDM-48, SDM-49, SDM-50, SDM-51,SDM-52, SDM-53, SDM-54, SDM-55, SDM-56, SDM-57, SDM-58, SDM-59, SDM-60,SDM-61, SDM-62, SDM-63, SDM-64, or SDM-65; and (b) visualizing at leastone of the imaging agents.

Disclosed herein, in certain embodiments, are methods of visualizing atissue of interest in an individual in need thereof, comprising:

(a) administering to the individual a molecule of Formula I thatlocalizes to the tissue of interest in the individual,

[c _(M)-M]-[[D_(A)-c _(A)]-(A-X-B)-[c _(B)-D_(B)]]  Formula I

-   -   wherein,        -   X is a cleavable linker;        -   A is a peptide with a sequence comprising 5 to 9 acidic            amino acids;        -   B is a peptide with a sequence comprising 7 to 9 basic amino            acids;        -   c_(A), c_(B), and c_(M) are independently 0-1 amino acid;        -   M is a polyethylene glycol (PEG) polymer; and        -   D_(A) and D_(B) are each independently an imaging agent; and    -   wherein [c_(M)-M] is bound at any position on or between A, X,        and B, [D_(A)-c_(A)] is bound to any amino acid on A or X, and        [c_(B)-D_(B)] is bound to any amino acid on B; and        (b) visualizing at least one of the imaging agents.        In some embodiments, the tissue is cancerous. In some        embodiments, the cancerous tissue is: breast cancer tissue,        colorectal cancer tissue, squamous cell carcinoma tissue, skin        cancer tissue, prostate cancer tissue, melanoma tissue, thyroid        cancer tissue, ovarian cancer tissue, or cancerous lymph node        tissue. In some embodiments, the cancerous cell or tissue is        breast cancer tissue. In some embodiments, the cancerous cell or        tissue is colorectal cancer tissue. In some embodiments, the        cancerous tissue is cancerous lymph node tissue. In some        embodiments, the cancerous tissue is squamous cell carcinoma        tissue. In some embodiments, the cancerous tissue is skin cancer        tissue. In some embodiments, the method further comprises        surgically removing the tissue of interest from the individual.        In some embodiments, the surgical margin surrounding the tissue        of interest is decreased. In some embodiments, the method        further comprises preparing a tissue sample from the removed        cell or tissue of interest. In some embodiments, the method        further comprises staging the cancerous tissue. In some        embodiments, A and B do not have an equal number of acidic and        basic amino acids. In some embodiments, the number of basic        amino acids in B is greater than the number of acidic amino        acids in A. In some embodiments, A is a peptide comprising 5 or        9 consecutive glutamates (SEQ ID NO: 4-5, respectively). In some        embodiments, B is a peptide comprising 8 or 9 consecutive        arginines (SEQ ID NO: 6-7, respectively). In some embodiments, A        is a peptide comprising 5 or 9 consecutive glutamates (SEQ ID        NO: 4-5, respectively) and B is a peptide comprising 8 or 9        consecutive arginines (SEQ ID NO: 6-7, respectively). In some        embodiments, A is a peptide comprising 5 consecutive glutamates        (SEQ ID NO: 4) and B is a peptide comprising 8 consecutive        arginines (SEQ ID NO: 6). In some embodiments, c_(A), c_(B), and        c_(m) are each independently a 0-1 amino acid. In some        embodiments, c_(A), c_(B), and c_(M) are each independently        selected from a naturally-occurring amino acid or a        non-naturally-occurring amino acid. In some embodiments, c_(A),        c_(B), and c_(M) are each independently selected from a D amino        acid, a L amino acid, an α-amino acid, a β-amino acid, or a        δ-amino acid. In some embodiments, c_(A), c_(B), and c_(M) are        each independently selected from any amino acid having a free        thiol group, any amino acid having a N-terminal amine group, and        any amino acid with a side chain capable of forming an oxime or        hydrazone bond upon reaction with a hydroxylamine or hydrazine        group. In some embodiments, c_(A), c_(B), and c_(M) are each        independently selected from D-cysteine, D-glutamate, lysine, and        para-4-acetyl L-phenylalanine. In some embodiments, c_(B) is any        amino acid having a free thiol group. In some embodiments, c_(B)        is D-cysteine. In some embodiments, c_(A) is any amino acid        having a N-terminal amine group. In some embodiments, c_(A) is        D-glutamate. In some embodiments, c_(A) is lysine. In some        embodiments, c_(M) is any amino acid with a side chain capable        of forming an oxime or hydrazone bond upon reaction with a        hydroxylamine or hydrazine group. In some embodiments, c_(M) is        para-4-acetyl L-phenylalanine. In some embodiments, X is        cleavable by a protease. In some embodiments, X is cleavable by        a matrix metalloproteinase. In some embodiments, X comprises an        amino acid sequence that is cleavable by MMP2, MMP7, MMP9, or        MMP14. In some embodiments, X comprises a peptide linkage. In        some embodiments, X comprises an amino acid sequence selected        from: PLGLAG (SEQ ID NO: 2), PLG-C(me)-AG (SEQ ID NO: 1),        RPLALWRS (SEQ ID NO: 3), ESPAYYTA (SEQ ID NO: 8), DPRSFL (SEQ ID        NO: 9), PPRSFL (SEQ ID NO: 10), RLQLKL (SEQ ID NO: 11), and        RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, X comprises the        amino acid sequence PLGLAG (SEQ ID NO: 2). In some embodiments,        X comprises the amino acid sequence PLG-C(me)-AG (SEQ ID NO: 1).        In some embodiments, X comprises the amino acid sequence        RPLALWRS (SEQ ID NO: 3). In some embodiments, X comprises the        amino acid sequence DPRSFL (SEQ ID NO: 9). In some embodiments,        X comprises the amino acid sequence PPRSFL (SEQ ID NO: 10). In        some embodiments, X comprises the amino acid sequence RLQLKL        (SEQ ID NO: 11). In some embodiments, X comprises the amino acid        sequence RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, D_(A)        and D_(B) are a pair of acceptor and donor fluorescent moieties        that are capable of undergoing Försters/fluorescence resonance        energy transfer with the other. In some embodiments, D_(A) and        D_(B) are Cy5 and Cy7. In some embodiments, D_(A) and D_(B) are        Cy5 and IRDye750. In some embodiments, D_(A) and D_(B) are Cy5        and IRDye800. In some embodiments, D_(A) and D_(B) are Cy5 and        ICG. In some embodiments, the method further comprises        visualizing Försters/fluorescence resonance energy transfer        between D_(A) and D_(B). In some embodiments, D_(A) and D_(B)        are a fluorescent moiety and a fluorescence-quenching moiety. In        some embodiments, the molecule of Formula I is: SDM-41, SDM-42,        SDM-43, SDM-44, SDM-45, SDM-46, SDM-47, SDM-48, SDM-49, SDM-50,        SDM-51, SDM-52, SDM-53, SDM-54, SDM-55, SDM-56, SDM-57, SDM-58,        SDM-59, SDM-60, SDM-61, SDM-62, SDM-63, SDM-64, or SDM-65. In        some embodiments, the molecule is SDM-41. In some embodiments,        the molecule of Formula I is SDM-42.

Disclosed herein, in certain embodiments, are methods of visualizing atissue of interest in an individual in need thereof, comprising,comprising (a) administering SDM-41 to the individual, and (b)visualizing at least one of the imaging agents.

Disclosed herein, in certain embodiments, are methods of visualizing atissue of interest in an individual in need thereof, comprising,comprising (a) administering SDM-42 to the individual, and (b)visualizing at least one of the imaging agents.

Disclosed herein, in certain embodiments, are methods of visualizing atissue of interest in an individual in need thereof, comprising,comprising (a) administering SDM-43 to the individual, and (b)visualizing at least one of the imaging agents.

Disclosed herein, in certain embodiments, are methods of visualizing atissue of interest in an individual in need thereof, comprising,comprising (a) administering SDM-44 to the individual, and (b)visualizing at least one of the imaging agents.

Disclosed herein, in certain embodiments, are methods of visualizing atissue of interest in an individual in need thereof, comprising,comprising (a) administering SDM-45 to the individual, and (b)visualizing at least one of the imaging agents.

Disclosed herein, in certain embodiments, are methods of visualizing atissue of interest in an individual in need thereof, comprising,comprising (a) administering SDM-46 to the individual, and (b)visualizing at least one of the imaging agents.

Disclosed herein, in certain embodiments, are methods of visualizing atissue of interest in an individual in need thereof, comprising,comprising (a) administering SDM-47 to the individual, and (b)visualizing at least one of the imaging agents.

Disclosed herein, in certain embodiments, are methods of visualizing atissue of interest in an individual in need thereof, comprising,comprising (a) administering SDM-48 to the individual, and (b)visualizing at least one of the imaging agents.

Disclosed herein, in certain embodiments, are methods of visualizing atissue of interest in an individual in need thereof, comprising,comprising (a) administering SDM-49 to the individual, and (b)visualizing at least one of the imaging agents.

Disclosed herein, in certain embodiments, are methods of visualizing atissue of interest in an individual in need thereof, comprising,comprising (a) administering SDM-50 to the individual, and (b)visualizing at least one of the imaging agents.

Disclosed herein, in certain embodiments, are methods of visualizing atissue of interest in an individual in need thereof, comprising,comprising (a) administering SDM-51 to the individual, and (b)visualizing at least one of the imaging agents.

Disclosed herein, in certain embodiments, are methods of visualizing atissue of interest in an individual in need thereof, comprising,comprising (a) administering SDM-52 to the individual, and (b)visualizing at least one of the imaging agents.

Disclosed herein, in certain embodiments, are methods of visualizing atissue of interest in an individual in need thereof, comprising,comprising (a) administering SDM-53 to the individual, and (b)visualizing at least one of the imaging agents.

Disclosed herein, in certain embodiments, are methods of visualizing atissue of interest in an individual in need thereof, comprising,comprising (a) administering SDM-54 to the individual, and (b)visualizing at least one of the imaging agents.

Disclosed herein, in certain embodiments, are methods of visualizing atissue of interest in an individual in need thereof, comprising,comprising (a) administering SDM-55 to the individual, and (b)visualizing at least one of the imaging agents.

Disclosed herein, in certain embodiments, are methods of visualizing atissue of interest in an individual in need thereof, comprising,comprising (a) administering SDM-56 to the individual, and (b)visualizing at least one of the imaging agents.

Disclosed herein, in certain embodiments, are methods of visualizing atissue of interest in an individual in need thereof, comprising,comprising (a) administering SDM-57 to the individual, and (b)visualizing at least one of the imaging agents.

Disclosed herein, in certain embodiments, are methods of visualizing atissue of interest in an individual in need thereof, comprising,comprising (a) administering SDM-58 to the individual, and (b)visualizing at least one of the imaging agents.

Disclosed herein, in certain embodiments, are methods of visualizing atissue of interest in an individual in need thereof, comprising,comprising (a) administering SDM-59 to the individual, and (b)visualizing at least one of the imaging agents.

Disclosed herein, in certain embodiments, are methods of visualizing atissue of interest in an individual in need thereof, comprising,comprising (a) administering SDM-60 to the individual, and (b)visualizing at least one of the imaging agents.

Disclosed herein, in certain embodiments, are methods of visualizing atissue of interest in an individual in need thereof, comprising,comprising (a) administering SDM-61 to the individual, and (b)visualizing at least one of the imaging agents.

Disclosed herein, in certain embodiments, are methods of visualizing atissue of interest in an individual in need thereof, comprising,comprising (a) administering SDM-62 to the individual, and (b)visualizing at least one of the imaging agents.

Disclosed herein, in certain embodiments, are methods of visualizing atissue of interest in an individual in need thereof, comprising,comprising (a) administering SDM-63 to the individual, and (b)visualizing at least one of the imaging agents.

Disclosed herein, in certain embodiments, are methods of visualizing atissue of interest in an individual in need thereof, comprising,comprising (a) administering SDM-64 to the individual, and (b)visualizing at least one of the imaging agents.

Disclosed herein, in certain embodiments, are methods of visualizing atissue of interest in an individual in need thereof, comprising,comprising (a) administering SDM-65 to the individual, and (b)visualizing at least one of the imaging agents.

In some embodiments, targeted delivery of an imaging agent to a cell ortissue enables a medical professional to visualize/image a specifictissue (e.g., cancerous tissue). In some embodiments, targeted deliveryof an imaging agent to a cell or tissue enables a medical professionalto remove (or, surgically excise) the tissue of interest (e.g.,cancerous tissue). In some embodiments, targeted delivery of an imagingagent to a cell or tissue enables a medical professional to remove (or,surgically excise) the tissue of interest (e.g., cancerous tissue) witha decrease in surgical margins. In some embodiments, targeted deliveryof an imaging agent to a cell or tissue enables a medical professionalto remove (or, surgically excise) a tumor/cancerous tissue and decreasesthe chance that some of the tumor/cancerous tissue will not be removed.In some embodiments, targeted delivery of an imaging agent to a cell ortissue enables a medical professional to maximally debulk atumor/cancerous tissue. In some embodiments, targeted delivery of animaging agent to cancerous breast tissue decreases the chances of anunnecessary operations and re-operations.

In some embodiments, targeted delivery of an imaging agent to a cell ortissue enables a medical professional to more accurately sample (e.g.,biopsy (e.g., excision biopsy, incision, biopsy, aspiration biopsy, orneedle biopsy)) tissue of interest (e.g., cancerous tissue). In someembodiments, targeted delivery of an imaging agent to a cell or tissueenables a medical professional to visualize/image a specific tissue(e.g., cancerous tissue) within an excised tissue containing healthytissue. Enabling identification of target tissue (e.g., canceroustissue) can guide the pathologist on where to section for pathologicalevaluation and decreases the chances of a pathologist missing unhealthytissue (e.g., cancerous tissue) and sampling healthy tissue which mayproduce a false negative. In some embodiments, tissue (e.g., canceroustissue) removed following use of a compound of Formula I is used toprepare a pathology section or slide. In some embodiments, canceroustissue removed following use of a compound of Formula I is used toprepare a pathology section or slide which is used to diagnose a tissueas malignant or benign.

In some embodiments, targeted delivery of an imaging agent to cancerousbreast tissue enables a medical professional to accurately stage cancerenabling medical treatment decisions. In some embodiments, targeteddelivery of an imaging agent to cancerous tissue enables a medicalprofessional to observe the size of a tumor (cancerous tissue) or thespread (e.g., metastatic lesions) of cancerous tissue. In someembodiments, targeted delivery of an imaging agent to a cell or tissueenables a medical professional to design an efficacious treatmentregimen.

In some embodiments, a selective delivery molecule according to FormulaI comprising an imaging agent is employed in guided surgery. In someembodiments, the selective delivery molecule preferentially localized tocancerous, or other pathological tissues with up-regulated proteaseactivity (e.g. tissues undergoing inflammatory response). In someembodiments, a selective delivery molecule according to Formula Icomprising an imaging agent is employed in a guided surgery to removecolorectal cancer. In some embodiments, guided surgery employing theselective delivery molecule allows a surgeon to excise as little healthy(i.e., non-cancerous) tissue as possible. In some embodiments, guidedsurgery employing the selective delivery molecule allows a surgeon tovisualize and excise more cancerous tissue than the surgeon would havebeen able to excise without the presence of the selective deliverymolecule. In some embodiments, the surgery is fluorescence-guidedsurgery.

Imaging Agents

In some embodiments, an imaging agent is a dye. In some embodiments, animaging agent is a fluorescent moiety. In some embodiments, afluorescent moiety is selected from: a fluorescent protein, afluorescent peptide, a fluorescent dye, a fluorescent material or acombination thereof.

All fluorescent moieties are encompassed within the term “fluorescentmoiety.” Specific examples of fluorescent moieties given herein areillustrative and are not meant to limit the fluorescent moieties for usewith the targeting molecules disclosed herein.

Examples of fluorescent dyes include, but are not limited to, xanthenes(e.g., rhodamines, rhodols and fluoresceins, and their derivatives);bimanes; coumarins and their derivatives (e.g., umbelliferone andaminomethyl coumarins); aromatic amines (e.g., dansyl; squarate dyes);benzofurans; fluorescent cyanines; indocarbocyanines; carbazoles;dicyanomethylene pyranes; polymethine; oxabenzanthrane; xanthene;pyrylium; carbostyl; perylene; acridone; quinacridone; rubrene;anthracene; coronene; phenanthrecene; pyrene; butadiene; stilbene;porphyrin; pthalocyanine; lanthanide metal chelate complexes; rare-earthmetal chelate complexes; and derivatives of such dyes.

Examples of fluorescein dyes include, but are not limited to,5-carboxyfluorescein, fluorescein-5-isothiocyanate,fluorescein-6-isothiocyanate and 6-carboxyfluorescein.

Examples of rhodamine dyes include, but are not limited to,tetramethylrhodamine-6-isothiocyanate, 5-carboxytetramethylrhodamine,5-carboxy rhodol derivatives, tetramethyl and tetraethyl rhodamine,diphenyldimethyl and diphenyldiethyl rhodamine, dinaphthyl rhodamine,rhodamine 101 sulfonyl chloride (sold under the tradename of TEXASRED®).

Examples of cyanine dyes include, but are not limited to, Cy3, Cy3B,Cy3.5, Cy5, Cy5.5, Cy7, IRDYE680, Alexa Fluor 750, IRDye800CW, ICG.

Examples of fluorescent peptides include GFP (Green Fluorescent Protein)or derivatives of GFP (e.g., EBFP, EBFP2, Azurite, mKalamal, ECFP,Cerulean, CyPet, YFP, Citrine, Venus, YPet).

Fluorescent labels are detected by any suitable method. For example, afluorescent label may be detected by exciting the fluorochrome with theappropriate wavelength of light and detecting the resultingfluorescence, e.g., by microscopy, visual inspection, via photographicfilm, by the use of electronic detectors such as charge coupled devices(CCDs), photomultipliers, etc.

In some embodiments, the imaging agent is labeled with apositron-emitting isotope (e.g., ¹⁸F) for positron emission tomography(PET), gamma-ray isotope (e.g., ^(99m)Tc) for single photon emissioncomputed tomography (SPECT), or a paramagnetic molecule or nanoparticle(e.g., Gd³⁺ chelate or coated magnetite nanoparticle) for magneticresonance imaging (MRI).

In some embodiments, the imaging agent is labeled with: a gadoliniumchelate, an iron oxide particle, a super paramagnetic iron oxideparticle, an ultra small paramagnetic particle, a manganese chelate orgallium containing agent.

Examples of gadolinium chelates include, but are not limited todiethylene triamine pentaacetic acid (DTPA),1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and1,4,7-triazacyclononane-N,N′,N″-triacetic acid (NOTA).

In some embodiments, the imaging agent is a near-infrared fluorophorefor near-infra red (near-IR) imaging, a luciferase (firefly, bacterial,or coelenterate) or other luminescent molecule for bioluminescenceimaging, or a perfluorocarbon-filled vesicle for ultrasound.

In some embodiments, the imaging agent is a nuclear probe. In someembodiments, the imaging agent is a SPECT or PET radionuclide probe. Insome embodiments, the radionuclide probe is selected from: a technetiumchelate, a copper chelate, a radioactive fluorine, a radioactive iodine,a indiuim chelate.

Examples of Tc chelates include, but are not limited to HYNIC, DTPA, andDOTA.

In some embodiments, the imaging agent contains a radioactive moiety,for example a radioactive isotope such as ²¹¹At, ¹³¹I, ¹²⁵I, ⁹⁰Y, ¹⁸⁶Re,¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, ³²P, ⁶⁴Cu radioactive isotopes of Lu, and others.

Starting Materials

Disclosed herein, in certain embodiments, are molecules of Formula II,having the structure:

A₁-X₁-B₁;  Formula II

wherein,

-   -   X₁ is a cleavable linker;    -   A₁ is a peptide with a sequence comprising 5 to 9 acidic amino        acids and having a first reactive amino acid moiety c_(A);    -   B₁ is a peptide with a sequence comprising 7 to 9 basic amino        acids and having a second reactive amino acid moiety c_(B); and    -   A₁-X₁-B₁ has a third reactive amino acid moiety c_(M) on A₁ or        X₁; and        wherein c_(A) is capable of reacting with a first cargo moiety        comprising D_(A), c_(B) is capable of reacting with a second        cargo moiety comprising D_(B), and c_(M) is capable of reacting        with a macromolecular carrier comprising M to form a molecule of        Formula I.        In some embodiments, the c_(A), c_(B), and c_(M) have functional        groups that are orthogonally reactive. In some embodiments,        c_(A), c_(B), and c_(M) are each independently selected from a        naturally-occurring amino acid or a non-naturally-occurring        amino acid. In some embodiments, c_(A), c_(B), and c_(M) are        each independently selected from a D amino acid, a L amino acid,        an α-amino acid, a β-amino acid, or a δ-amino acid. In some        embodiments, c_(A), c_(B), and c_(M) are each independently        selected from any amino acid having a free thiol group, any        amino acid having a N-terminal amine group, and any amino acid        with a side chain capable of forming an oxime or hydrazone bond        upon reaction with a hydroxylamine or hydrazine group. In some        embodiments, c_(A), c_(B), and c_(M) are each independently        selected from D-cysteine, D-glutamate, lysine, and para-4-acetyl        L-phenylalanine. In some embodiments, c_(B) is any amino acid        having a free thiol group. In some embodiments, c_(B) is        D-cysteine. In some embodiments, c_(A) is any amino acid having        a N-terminal amine group. In some embodiments, c_(A) is        D-glutamate. In some embodiments, c_(A) is lysine. In some        embodiments, c_(M) is any amino acid with a side chain capable        of forming an oxime or hydrazone bond upon reaction with a        hydroxylamine or hydrazine group. In some embodiments, c_(M) is        para-4-acetyl L-phenylalanine.

As used herein, “orthogonally reactive” means a plurality of groups canbe attached to a molecule via a sequence of reactions that do not crossreact enabling specific attachment of each group in the presence of theothers. In some embodiments, the three groups (D_(A), D_(B), and D_(M))are able to be attached to A₁-X₁-B₁ via c_(A), c_(B), and c_(M) using asequence of 3 independent reactions that do not cross react so that eachgroup is attached to only one site on A₁-X₁-B₁.

Disclosed herein, in certain embodiments, is a molecule having the aminoacid sequence:

-   -   (D-Glu)₅-F(4-Ac)-o-Pro-Leu-Gly-Cys(_(Me))-Ala-Gly-(D-Arg)₈-(D-Cys)        wherein o represent 5-(amino-3-oxapentanoyl); F_((4-Ac))        represent para-acetyl-(L)-phenylalanine; and C_((Me)) represents        S-methyl-(L)-cysteine.

In some embodiments, the molecule further comprises a polyethyleneglycol (PEG) polymer. In some embodiments, the PEG polymer is covalentlylinked to the molecule at the F(4-Ac) subunit. In some embodiments, themolecule comprises groups that can be orthogonally reacted. In someembodiments, the groups that can be orthogonally reacted are chosenfrom: an amine, thiol and an acetyl phenylalanine. In some embodiments,the molecule comprises an amine, a thiol, and an acetyl phenylalanine.

In some embodiments, the PEG polymer has an average molecular weight ofapproximately 0.5 kDa. In some embodiments, the PEG polymer has anaverage molecular weight of approximately 2 kDa. In some embodiments,the PEG polymer has an average molecular weight of approximately 5 kDa.In some embodiments, the PEG polymer has an average molecular weight ofapproximately 10 kDa. In some embodiments, the PEG polymer has anaverage molecular weight of approximately 20 kDa. In some embodiments,the PEG polymer has an average molecular weight of approximately 40 kDa.Disclosed herein, in certain embodiments, is the use of the molecule inthe synthesis of a molecule according to Formula I.

Disclosed herein, in certain embodiments, is a molecule having the aminoacid sequence:

-   -   (D-Glu)₅-o-Pro-Leu-Glys-Cys(_(me))-Ala-Gly-(D-Arg)₈-(D-Cys)-[PEG_((2k))]        wherein all glutamates and arginines are D-amino acids; o        represents 5-(amino-3-oxapentanoyl); Cys(_(me)) represents        S-methyl-(L)-cysteine; and PEG(_(2k)) represents α-amino-ω-amide        poly(ethylene glycol) with an average molecular weight of        approximately two kDa. In some embodiments, the molecule further        comprises a fluorescent moiety. Disclosed herein, in certain        embodiments, is the use of the molecule in the synthesis of a        molecule according to Formula I.

EXAMPLES Materials and Methods

HPLC-grade acetonitrile, glycine, acetophenone and aniline werepurchased from Thermo Fisher Scientific (Waltham, Mass.). Purified waterwas collected through Milli-Q water purification system (Millipore,Bedford, Mass.). 3-Maleimidopropionic acid-Pfp ester was purchased fromMolecular Biosciences (Boulder, Colo.). PBS-EDTA buffer was purchasedfrom Teknova (Hollister, CA). PBS buffer (pH 8.5, 0.5 M) was purchasedfrom Boston Bioproducts (Ashland, Mass.). Trifluoroacetic acid (TFA) waspurchased from Alfa Aesar (Ward Hill, Mass.). Dimethylformamide (DMF)and N-methylmorpholine (NMM) were supplied by Sigma-Aldrich (Milwaukee,Wis.). α-Mercaptoethyl-ω-methoxy, poly-oxyethylene (Mw ˜2,000, ˜5,000,˜20,000 and ˜40,000) [mPEG(2K)-SH, mPEG(5K)-SH, mPEG(20K)-SH,mPEG(40K)-SH] and α-aminoxyl-ω-methoxy, polyoxyethylene (Mw ˜2,000,˜5,000, ˜10,000, ˜20,000 and ˜40,000) [mPEG(2K)-ONH₂, mPEG(5K)-ONH₂,mPEG (10K)-ONH₂, mPEG(20K)-ONH₂, mPEG(40K)-ONH₂] were purchased from NOFAmerica Corporation (Irvine, Calif.). mPEG(1K)-NHNH₂ (Mw ˜1,000) waspurchased from Nanocs (New York). IRDye 800CW maleimide (Mal-IRDye) andIRDye 750 succinimidyl ester were supplied by Li-Cor Biosciences(Lincoln, Nebr.). Lyophilized peptides P-1 to P-18 were supplied byPolyPeptide Group (San Diego, Calif.).

LC-MS analysis was carried out on an Agilent 1200 SL series incombination with AB SCIEX API 3200, equipped with CTC PAL autosampleroperating at 4° C., a vacuum degasser, binary pump, UV-VIS detector,associated Analyst 1.5 analytical software and a Phenomenex column(Kinetex 2.6μ C18 100A, 100×2.1 mm) or a Waters 2695 separation moduleequipped with a Waters 2487 dual X absorbance detector in combinationwith Finnigan LCQ Deca XP mass spectrometer. The equipment is associatedwith Xcalibur analytical software and Peeke Scientific columns (Titan200 5 μm, C18-MC, 50/100×2.1 mm).

Preparation HPLC were carried out on an Agilent system (Agilent 1200series) and a Thermo Scientific column (Hypersil Gold C18, 5μ, 250×10mm), or a Waters Delta Prep preparative HPLC System and a Varian column(F75L, C18, 15μ, 1200 g), or a Waters PrepLC System equipped with aWaters 2487 dual λ absorbance detector, Fraction Collector III, Masslynxsoftware and a Thermo Scientific column (Hypersil Gold C18, 5μ, 250×10mm) or a Phenomenex column (luna, C18(2), 5μ, 100 A AX 150×30 mm). Themobile phase consisted of a water (0.05% TFA)(solvent A)/acetonitrile(0.05% TFA)(solvent B) gradient.

Centrifugation was carried out at 4° C. with an Eppendorf centrifuge5810R or a Beckman Microfuge® 18.

Exemplary materials for synthesis of the selective delivery moleculesdisclosed herein include, but are not limited to, any of peptides P-1,P-2, P-3, P-4, P-5, P-6, P-7, P-8, P-9, P-10, P-11, P12, P-13, P-14,P-15, P-16, P-17 and P-18.

The above starting materials are summarized below:

Peptide Sequences Peptide P-1 eeeeeeeeeoPLGC_((Me))AGrrrrrrrrrcPeptide P-2 eeeeeoPLGC_((Me))AGrrrrrrrrc Peptide P-3eeeeeF_((4-Ac))oPLGC_((Me))AGrrrrrrrrc Peptide P-4eeeeeeeeeF_((4-Ac))oPLGC_((Me))AGrrrrrrrrrc Peptide P-5(Ac)eeeeeoPLGC_((Me))AGrrrrrrrrck Peptide P-6eeeeeoPLGC_((Me))AGoF_((4-Ac))rrrrrrrrc Peptide P-7eeeeeeeeeoPLGC_((Me))AGrrrrrrrrrcoF_((4-Ac)) Peptide P-8[mPEG_((2K))]crrrrrrrrPLGC_((Me))AGoeeeeek Peptide P-9[mPEG_((5K))]crrrrrrrrPLGC_((Me))AGoeeeeek Peptide P-10eeeeeoPLGC_((Me))AGrrrrrrrrc[PEG_((3K))] Peptide P-11(Aeo)F_((4-Ac))oPLGC_((Me))AG(Aeo)(Aeo)c Peptide P-12eeeee F_((4-Ac))(Aeo)(Aeo)rrrrrrrrc Peptide P-13eeeeeF_((4-Ac))oPLGC_((Me))AGrrrrrrrwc Peptide P-14(Ac)keeeeeF_((4-Ac))oPLGC_((Me))AGrrrrrrrrc Peptide P-15oeeeeeF_((4-Ac))oPLGC_((Me))AGrrrrrrrrc Peptide P-16eeeeeF_((4-Ac))oRPLALWRSrrrrrrrrc Peptide P-17eeeeeeeeeF_((4-Ac))oRPLALWRSrrrrrrrrrc Peptide P-18[mPEG_((2K))]eeeeekoPLGC_((Me))AGrrrrrrrrc Abbreviations: Standard 1letter amino acid abbreviations were used in all the sequences.Lowercase characters indicated D-amino acids. All peptides were amidatedat C-terminus. o: 5-(amino-3-oxapentanoyl); F_((4-Ac)):para-acetyl-(L)-phenylalanine; C_((Me)): S-methyl-(L)-cysteine. PEG(3k):α-amino-ω-amide poly(ethylene glycol) with an averaged three thousandDaltons molecular weight; mPEG(2k): α-carboxy-ω-methoxy poly(ethyleneglycol) with an averaged two thousand Daltons molecular weight;mPEG(5k): α-carboxy-ω-methoxy poly(ethylene glycol) with an averagedfive thousand Daltons molecular weight. Ac: acetyl (Aeo):2-(2-2(aminoethoxy)ethoxy)acetyl

Example 1: Synthesis of SDM-41 from Peptide P-16

Synthesis of Intermediate 2

To a solution of peptide P-16 (10 mg, 2.0 μmol) in acetonitrile (0.5 mL)and PBS buffer (0.5 mL, pH 8.5, 0.5 M) at room temperature in the darkwas added Cy5 maleimide (2.3 mg, 2.7 μmol) with stirring. The reactionwas followed by LC-MS and completed in 40 min. To the reaction mixturewas added Cy7-NHS followed by PBS buffer (1.0 mL, pH 8.5, 0.5 M). Afterstirring for 15 h, the mixture was purified by HPLC to affordintermediate 2 (3.8 mg, 32%). Calculated: [M+3H]³⁺ (C₂₁₁H₃₁₉N₆₂O₅₃S₅)m/z=1577; Found ESI: [M+3H]³⁺ (C₂₁₁H₃₁₉N₆₂O₅₃S₅) m/z=1577.

Synthesis of Selective Delivery Molecule SDM-41

The mixture of intermediate 2 (3.8 mg, 0.65 μmol) and mPEG(2K)-ONH₂ (2.5mg, 1.1 μmol) in glycine buffer (1.0 mL, 0.1 M, 20 mM aniline, pH 3.0)and acetonitrile (0.5 mL) was stirred at room temperature in the darkfor 15 h. After the reaction was complete, acetophenone (7 μL, 60 μmol)was added. The mixture was stirred at room temperature for 2 h.Purification by RP-HPLC afforded selective delivery molecule SDM-41 (2.1mg, 40%).

Example 2a: Enzyme Dependent Fluorescence Enhancement and Color Changes

Selective delivery molecule 41 was dissolved in TCNB buffer (pH 7.5) atroom temperature. Concentration of SDM-41 was 0.156 to 5 μM.Fluorescence intensity was measured on a Molecular Devices Spectromax M2spectrophotometer. The sample was excited at 620 nm and the emission wasmeasured at 670 nm (Cy5).

Peptide cleavage was initiated with addition of MMP-7 at a. finalconcentration of 1 nM. Cleavage rate was measured as change in relativefluorescence intensity of Cy5 per minute, which was subsequentlyconverted to concentration of Cy5-peptide with a standard curve in orderto calculate k_(cat) and K_(m) for cleavage of SDM-41 b (FIG. 1). Thedata show that SDM-41 is substrate for MMP-7 and that it generates afluorogenic FRET signal upon enzyme cleavage.

Example 2b: Enzyme Dependent Fluorescence Enhancement and Color Changes

Selective delivery molecule 42 is dissolved in TCNB buffer (pH 7.5) atroom temperature at 1 μM. Fluorescence spectra are recorded on F-2500fluorescence spectrometer. Excitation of the Cy5 fluorescence donor isexcited at 625 nm and the emission is measured at 669 nm.

Peptide cleavage is initiated with addition of MMP-2 at a finalconcentration of 1 nM. The cleavage reaction is complete within 2 hours.

Example 3: Fluorogenic Response from Tumor Homogenates

HT1080 cells (Cat. #CCL-121; American Type Culture Collection, VA, USA)are grown under exponential growth conditions in humidified atmosphereof 5% CO₂ in air at 37° C. until reaching 80-100% confluence beforeharvesting for mouse implantation. Each nude mouse is hand restrainedand injected with 2×10⁶ HT-1080 cells into the mammary fat pad using a25-G needle. HT-1080 tumors are harvested when they had reached 100-200mm³ in size (typically 1-2 weeks post-tumor cells implantation).

HT-1080 tumors are homogenized using ultrasonic disruption. 1 nM MMP-7or 10 μL tumor tissue homogenates (TH2 and TH3) are mixed with 1 μMSDM-42 in 100 μL buffer for 24 h at 37° C. The samples are loaded on apolyacrylamide gel and separated using electrophoresis. After incubationwith HT-1080 tumor homogenates, SDM-42 is cleaved and becomes highlyfluorescent.

Example 4: In Vivo Imaging Assay for Tumor Contrast

HT-1080 xenograft model is generated as described in Example 3 and usedto evaluate the ability of molecules to provide in vivo tumorfluorescence contrast compared to surrounding tissue. Fluorescentconjugates are tested in HT-1080 tumor-bearing mice once the tumors hadreached 100-200 mm³ in size (typically 1-2 weeks post-tumor cellsimplantation). Conscious HT-1080 tumor-bearing mice are restrained usinga rotating tail injector (Cat. #RTI; Braintree Scientific, MA, USA) anddosed intravenously (tail vein) with SDM-43, SDM-44, SDM-45 or SDM-46 atbetween 0.1 and 5 nanomoles per mouse in 100 uL saline solution. Inpreparation for imaging, mice are lightly anesthetized with a mixture ofketamine/xylazine (Cat. #K-113; Sigma, Aldrich, MO, USA) givenintraperitoneally (1 μL/gram body weight) to minimize movement.

Serial whole-body imaging (tumor included) is done using a whole-animalfluorescent visualization imaging system or Olympus stereo fluorescentmicroscope. The mice are positioned on their backs and imaging isperformed from the top to image the ventral side of the animal.Excitation and emission wavelengths are selected based on thefluorescent dye used. Contrast is calculated using the followingequation:

Contrast=(Fluorescence intensity of tumor −Fluorescence intensity ofcontralateral chest tissue)/Intensity of contralateral chest tissue).

Contrast greater than 0.4 in the whole animal is easily detected by eyein the whole animal image and is good contrast. Contrast >0.7 is highcontrast.

The mice are imaged several times between 1-24 hours after injection.

Example 5: In Vivo Distribution and Compounds with Improved TissueAccumulation

To determine the total dye accumulation in various organs, HT-1080xenograft mice are sacrificed and tissue samples from blood, liver,kidney, and tumor are collected 6 hours after compounds are administerediv via the tail vein. 3-4 mice are used for each data point. Bloodsamples are stored at 4° C. overnight and then centrifuged at 15,000 rpmto separate out the serum. The organs are mixed in a ProK buffer (0.25mg/ml Prok, 0.1 mg/ml DNAse, 150 mM NaCl, 10 mM Tris pH8.0, 0.2% SDS) at10 μL/mg tissue and cut into small pieces using scissors. Thetissue/digest solution is then sonicated for 1 minute at 67% duty cycleand digested overnight at 37° C. After digestion, the sample iscentrifuged at 15,000 rpm and the tissue homogenate is aspirated off andstored at 4° C.

The tissue concentration of fluorescent compounds is determined fromfluorescence standard curves generated by spiking in know concentrationsof administered compounds into serum and tissue homogenates (at variousdilutions) from control animals that are not injected with compound. Thelinear range for each compound is determined for each tissue.Fluorescence measurements are done on either a fluorescent plate readeror fluorescence spectrometer.

Example 6a: In Vivo Detection of Cancer Metastases to Lymph Node withFRET SDMs

Fluorescence labeling of metastatic cervical lymph nodes followingintravenous and peritumoral administration of fluorescent SDMs in tumorbearing mice.

The following model and assays were used to determine the ability offluorescent SDMs to detect cancer metastases to lymph nodes inimmunocompetent BALB/c mice (Charles River, Wilmington, Mass. 01887)bearing syngeneic ear tumors.

Mouse Model. The mice were housed in groups of 4 in individuallyventilated IVC disposable cages (Innovive, Inc., San Diego, Calif.92121) and had free access to standard laboratory chow (Cat. #2018,Harlan Laboratories, Inc. Indianapolis, Ind. 46250) and drinking water.Animals were kept under controlled environmental conditions (12-h/12-hlight/dark cycle) for at least 5 days before tumor cell implantation.All experimental procedures were carried out under the approved IACUCprotocol #EB11-002-009A. Murine 4T1 tumor (ATCC® Number: CRL-2539™) andmammary carcinoma (Polyoma Middle T 8119 subclone “PyMT 8119”) cellsfrom the American Type Culture Collection (ATCC, Manassas, Va. 20108)and the University of San Diego, Calif. (UCSD, La Jolla, Calif. 92093)respectively were grown separately using standard cell culturetechniques. Tumor cells (4×10⁵ tumor cells/50 μL/mouse) were suspendedin DPBS/Matrigel™ (1:1 vol) and injected subcutaneously on the mouse earpinna above the auricular cartilage for primary tumor induction. The invivo imaging of metastatic cervical lymph nodes in ear tumor-bearingmice used as surrogate murine model of metastatic breast cancer tookplace seventeen to twenty days following tumor cell implantation.

Compound administration. For the intravenous administration (tail veininjection) of SDMs, mice were restrained in a rotating tail injector(Cat. #RTI, Braintree Scientific, Inc., Braintree, Mass. 02185) and thetest article (5-120 04; 100 μL/mouse) injected in mouse using a28G^(1/2) insulin syringe (Cat. #14-826-79, Becton Dickinson andCompany, Franklin Lakes, N.J. 07417). To perform the peritumoralinjection of SDMs, each involved mouse was sedated using theketamine/xylazine (Ketaject® & Xyla-ject®, Phoenix Pharmaceuticals, St.Joseph, Mo. 64506) mixture administered intraperitoneally and the testarticle (5-120 04; 30-60 μL/ear) injected subcutaneously around theprimary tumor and contralateral ear pinna using a 30G PrecisionGlide™needle (Cat. #305106, Becton Dickinson and Company, Franklin Lakes, N.J.07417). After dosing, each mouse was returned to the assigned cage andkept under controlled environmental conditions before being examined forthe fluorescence imaging of cervical lymph nodes 1-24 hours later.

Fluorescence imaging. To image the cervical lymph nodes, each mouse wasdeeply anesthetized with a mixture of ketamine/xylazine administeredintraperitoneally. The deeply anesthetized mouse was transferred on apiece of black cork (4×4 inches, Quartet®, ACCO Brands, Lincolnshire,Ill. 60069, USA) for blunt dissection and imaging of cervical lymphnodes using a computerized fluorescent stereomicroscope (SZX10, OlympusOptical, CO, LTD, Japan) equipped with appropriate fluorescence filtersfor both single intensity and two fluorophore fluorescence ratiodetection. For example, filters for Cy5 and Cy7 were used for FRET-basedSDMs with Cy5 and Cy7. After in vivo fluorescence imaging (see below forratio imaging method), the cervical lymph nodes were surgically removed,fixed in 10% buffered formalin and processed for histology (Hematoxylin& Eosin staining) to assess the fluorescence/cancer correlation anddetermine diagnostic performance of SDMs.

Emission Ratio Imaging Method. Fluorescence images were acquired usingan Olympus SZX10 Research Stereo Microscope (Olympus America, CenterValley, Pa.). For Cy5 and Cy7 FRET-based SDMs an excitation filtercentered at 620 nm (Chroma ET620/60x, Chroma Technology Corp. BellowsFalls, Vt.) and emission filters centered at 700 nm and 810 nm (Chromafilters ET700/75m and ET810/90m) were used to produce two images atdifferent emission wavelengths. Images were acquired with an Orca-R2camera (Hamamatsu, Bridgewater, N.J.) connected to a Windows-basedcomputer. Two methods were used to determine emission ratios for lymphnodes. For one method the intensity was averaged over a region ofinterest (ROI) drawn to include part or all of the lymph node ofinterest. The Emission ratio was then calculated from the intensity datafor each region of interest.

Roi EmissionRatio=(roiInt1/Exp1)/(Int2/Exp2)  (equation 1)

-   -   where:    -   roiInt1=averaged intensity for ROI at emission wavelength 1 with        ET700/75m filter    -   Exp1=exposure time used for Int1    -   roiInt 2=average intensity for ROI at emission wavelength 2 with        ET810/90m filter    -   Exp 2=exposure time used for Int2

A second method used to determine emission ratios was based averagingthe emission ratio from a region of interest (ROI) drawn to include partor all of the lymph node of interest taken from an emission ratio image.Emission ratio images were produced by using a modified form of equation1 that included a scaling factor so that the pixel values would fallbetween 0 and 255 for an 8-bit image.

Px EmissionRatio=k*(pxInt1/Exp1)/(pxInt2/Exp2)  (equation 2)

where:

-   -   k=scaling factor    -   pxInt1=pixel intensity at emission wavelength 1 with ET700/75m        filter    -   Exp1=exposure time used for Int1    -   pxInt 2=pixel intensity at emission wavelength 2 with ET810/90m        filter    -   Exp 2=exposure time used for Int2

An example of an emission ratio images generated using equation 2 whereExp1=0.7 sec, Exp2=2.5 sec and k=24 for SDM-41 is shown in FIG. 2, whichshows the donor (left), acceptor (middle) and fluorescence emissionratio (right) images for SDM-41.

Emission ratios for lymph nodes gave quantitatively similar resultsusing either method.

Lymph nodes were identified as either metastatic or non-metastatic by apathologist based on H&E staining. Emission ratio contrast for each SDM(selective delivery molecule) was then quantified by dividing theaverage emission ratio of the metastatic nodes by the average emissionof the non-metastatic nodes and subtracting one as shown in equation 3:

ERC=MetAV/ConAV−1  (equation 3)

where:

-   -   ERC=emission ratio contrast    -   MetAV=average metastatic lymph node emission ratio    -   ConAV=average non-metastatic contralateral lymph node emission        ratio

Although useful for detecting cancerous lymph nodes, a contrast of 20 to50% was considered low, an increase of 50 to 100% was considered good,while an increase greater than 100% was considered excellent.

Example 6b: High Diagnostic Sensitivity and Specificity for SDM inMetastatic Lymph Node Model

Key performance metrics of a diagnostic agent are sensitivity andspecificity. Sensitivity relates to the ability to correctly diagnosetest positives. While specificity relates to the ability to correctlydiagnose test negatives.

The following model and assays were used to determine the ability offluorescent SDMs to detect cancer metastases to lymph nodes inimmunocompetent BALB/c mice (Charles River, Wilmington, Mass. 01887)bearing syngeneic ear tumors.

As an example of high diagnostic performance of a FRET SDM, datagenerated from SDM-41 in the 4T1 mouse metastatic lymph model was used.SDM-41 was administered via IV tail vein injection. After 3 to 6 hours,the mice lymph nodes were imaged using fluorescence ratio imaging asdescribed previously to determine whether or not the lymph node had ahigh ratio (diagnosed cancer positive) or low ratio (diagnosed cancernegative). Sensitivity and specificity was determined using receiveroperating characteristic (ROC) or ROC curves. For ROC curve analysis,data is divided into a binary classification of positives and negativesbased on a threshold value for the emission ratio. The ROC curve plotstrue positive fraction of positives (true positive rate) versus falsepositive fraction of negatives (false positive rate).

True positives, false positives, true negatives, and false negativeswere determined by comparing the prediction based on the fluorescenceemission ratio data and threshold value with the positive or negativeassignment made by a pathologist using H&E staining. The emission ratiovalues for the cancer positive and negatives (as determined by H&Estaining by a certified pathologist) are shown in FIG. 3. The twopopulations are completely separated demonstrating 100% diagnosticsensitivity and specificity in this metastatic breast cancer lymph nodemodel. The threshold value was gradually adjusted from low to high toobtain a full ROC curve from (1, 1) or all positives to (0, 0) or allnegatives. A ROC curve is shown in FIG. 4. Data from ˜32 lymph nodeswere used to generate this curve. Note that sensitivity and specificitycan be determined for each point in the ROC curve. This data illustrates100% diagnostic sensitivity and specificity for separating cancerouslymph nodes from those without cancer. Sensitivity is the true positiverate while specificity is one minus the false positive rate. Equationsused to generate the ROC curve are shown below.

TPR=TP/(TP+FN)

FPR=FP/(FP+TN)

where:

-   -   TPR=true positive rate    -   FPR=false positive rate    -   TP=# of true positives    -   TN=# of true negatives    -   FP=# of false positives    -   FN=# of false negatives

In this example both sensitivity and specificity are 100% for allthreshold values between ˜4.3 and ˜5. This means that all lymph nodeswere correctly identified with the FRET emission ratio method whencompared to the gold standard histopathology. Generally, sensitivity andspecificity values >90% are considered very high.

Example 7: Ex Vivo Mouse PyMT 8119 Tumor Activity Assay: SDM Cleavageand FRET Emission Ratio Response in Mouse Cancer Tissue Compared to NonCancerous Tissue

Tumor and muscle tissue samples from PyMT 8119 tumor bearing mice arecollected and frozen at −80° C. The tissues are thawed and homogenizedin cold TCNB buffer (pH 7.5, 50 mM Tris-HCl, 10 mM CaCl₂), 150 mM NaCland 0.05% Brij35) at 100 mg/200 μL, using ultrasonic disruption (VCX500,Sonics & Materials Inc, Newtown, Conn.). After homogenates arecentrifuged at 15,000 g at 4° C. for 20 min, supernatants are collected.APMA (p-aminophenylmercuric acetate, 90 μL, 2 mM in TCNB buffer) isadded to the supernatants (90 μL). The resulting mixtures are incubatedat 37° C. for 1 h before use. 500 nM of SDM-42 is used for the cleavageof 45 μL, of activated tissue supernatants (final volume: 50 μL). Theassay is carried out using a SpectraMax M2 spectrometer with SoftMax Prov4.5 software. Fluorescence signals of (λex, 620 nm, λem, 670 nm), (λex,620 nm, λem, 773 nm) and (λex, 720 nm; λem, 773 nm), where λex and λemstand for excitation and emission wavelengths respectively, are measuredas a function of time at room temperature. Samples are measured intriplicate.

Example 8: Human Ex Vivo Tissue Assay: SDM Cleavage and FRET EmissionRatio Response in Human Cancer Tissue Compared to Noncancerous Tissue

Human breast cancer tissue samples and normal human breast tissue(provided by Cancer Human Tissue Network) were homogenized in cold TCNBbuffer (pH 7.5, 50 mM Tris-HCl, 10 mM CaCl₂), 150 mM NaCl and 0.05%Brij35) at 100 mg/200 μL, using ultrasonic disruption (VCX500, Sonics &Materials Inc, Newtown, Conn.). After homogenates were centrifuged at15,000 g at 4° C. for 20 min, supernatants were collected. 500 nM ofSDM-41 was used for the cleavage of 45 uL of tissue supernatant (finalvolume: 50 μL) in the assay unless otherwise noted. The assay wascarried out using a SpectraMax M2 spectrometer with SoftMax Pro v4.5software. Fluorescence signals of (λex, 620 nm, λem, 670 nm), (λex, 620nm, λem, 773 nm) and (λex, 720 nm; λem, 773 nm), where λex and λem standfor excitation and emission wavelengths respectively, were measured as afunction of time at room temperature. Samples were measured intriplicate. Table 1 shows the human breast cancer patient tissue usedfor the SDM-41 diagnostic fluorescence ex vivo assay. FIG. 5 shows thechange in SDM-41 fluorescence ratio in homogenized cancerous and healthytissue from breast cancer patients. The cancerous tissue (M112090A2,M1121603A2, and M1121797A6) cleaves SDM-41 faster than normal tissue((M112090B2, M1121603B2, and M1121797B6) and enables diagnostic readoutof cancerous breast cancer tissue. FIG. 6 shows the change in SDM-41fluorescence ratio in homogenized cancerous and healthy tissue from thesame breast cancer patients. The cancerous tissue cleaves SDM-41 fasterand enables diagnostic readout of cancerous breast cancer tissue.

TABLE 1 Age/Gender/Race Diagnosis Sample ID Patient 1 63/female/whitePleomorphic Tumor: M1120909A2 lobular carcinoma Normal: M1120909B2Patient 2 69/female/white Invasive ductal Tumor: M1121797A6 carcinomaNormal: M1121797B6 Patient 3 69/female/white Invasive ductal Tumor:M1121603A2 carcinoma Normal: M1121603B2

Example 9: Use of an SDM to Visualize Cancer in Breast Cancer Patients

SDM-52 is delivered intravenously to a breast cancer patient. Thefluorescent moieties on SDM-52 are taken up by cancerous cells and/ortissue after cleavage of the linker. A light source is shined onto thetarget tissue. The fluorescent moieties emit light which is detected bya camera or a detector. The data obtained by the camera or detector isprocessed to generate an image that allows the surgeon to visualizecancerous cells or tissue. The surgeon excises said tissue for biopsy.

Example 10: Use of an SDM to Visualize Cancer in Prostate CancerPatients

SDM-42 is delivered intravenously to a prostate cancer patient. Thefluorescent moieties on SDM-42 are taken up by cancerous cells and/ortissue after cleavage of the linker. A light source is shined onto thetarget tissue. The fluorescent moieties emit light which is detected bya camera or a detector. The data obtained by the camera or detector isprocessed to generate an image that allows the surgeon to visualizecancerous cells or tissue. The surgeon excises said tissue for biopsy.

Example 11: Use of an SDM to Visualize Cancer in Patients with Head andNeck (Squamous) Cancer

SDM-48 is delivered intravenously to a head and neck cancer patient. Thefluorescent moieties on SDM-48 are taken up by cancerous cells and/ortissue after cleavage of the linker. A light source is shined onto thetarget tissue. The fluorescent moieties emit light which is detected bya camera or a detector. The data obtained by the camera or detector isprocessed to generate an image that allows the surgeon to visualizecancerous cells or tissue. The surgeon excises said tissue for biopsy.

Example 12: Use of an SDM to Visualize Cancer in Patients with Melanoma

SDM-60 is delivered intravenously to a patient having melanoma. Thefluorescent moieties on SDM-60 are taken up by cancerous cells and/ortissue after cleavage of the linker. A light source is shined onto thetarget tissue. The fluorescent moieties emit light which is detected bya camera or a detector. The data obtained by the camera or detector isprocessed to generate an image that allows the surgeon to visualizecancerous cells or tissue. The surgeon excises said tissue for biopsy.

Example 13: Use of an SDM to Visualize Cancer in Patients with ThyroidCancer

SDM-62 is delivered intravenously to a thyroid cancer patient. Thefluorescent moieties on SDM-62 are taken up by cancerous cells and/ortissue after cleavage of the linker. A light source is shined onto thetarget tissue. The fluorescent moieties emit light which is detected bya camera or a detector. The data obtained by the camera or detector isprocessed to generate an image that allows the surgeon to visualizecancerous cells or tissue. The surgeon excises said tissue for biopsy.

What is claimed is:
 1. A selective delivery molecule having thestructure of SDM-44:


2. A method of delivering Cy5 and Cy7 to a tissue of interest,comprising contacting the tissue of interest with a selective deliverymolecule of claim
 1. 3. The method of claim 2, wherein the tissue ofinterest is cancerous.
 4. The method of claim 3, wherein the canceroustissue is: breast cancer tissue, colorectal cancer tissue, squamous cellcarcinoma tissue, skin cancer tissue, prostate cancer tissue, melanomatissue, thyroid cancer tissue, ovarian cancer tissue, cancerous lymphnode tissue, cervical cancer tissue, lung cancer tissue, pancreaticcancer tissue, head and neck cancer tissue, or esophageal cancer tissue.5. The method of claim 3, wherein the cancerous tissue is breast cancertissue.
 6. The method of claim 3, wherein the cancerous tissue iscolorectal cancer tissue.
 7. The tissue sample of claim 3, wherein thecancerous tissue is squamous cell carcinoma tissue.
 8. The tissue sampleof claim 3, wherein the cancerous tissue is skin cancer tissue.
 9. Thetissue sample of claim 3, wherein the cancerous tissue is cancerouslymph node tissue.
 10. A method of visualizing a tissue of interest inan individual in need thereof, comprising: (a) administering to theindividual a selective delivery molecule of claim 1; and (b) visualizingat least one of Cy5 or Cy7 of the selective delivery molecule.
 11. Themethod of claim 10, wherein the cancerous tissue is: breast cancertissue, colorectal cancer tissue, squamous cell carcinoma tissue, skincancer tissue, prostate cancer tissue, melanoma tissue, thyroid cancertissue, ovarian cancer tissue, cancerous lymph node tissue, cervicalcancer tissue, lung cancer tissue, pancreatic cancer tissue, head andneck cancer tissue, or esophageal cancer tissue.
 12. The method of claim11, wherein the cancerous tissue is breast cancer tissue.
 13. The methodof claim 11, wherein the cancerous tissue is colorectal cancer tissue.14. The tissue sample of claim 11, wherein the cancerous tissue issquamous cell carcinoma tissue.
 15. The tissue sample of claim 11,wherein the cancerous tissue is skin cancer tissue.
 16. The tissuesample of claim 11, wherein the cancerous tissue is cancerous lymph nodetissue.
 17. The method of claim 11, further comprising surgicallyremoving the tissue of interest from the individual.
 18. The method ofclaim 17, further comprising preparing a tissue sample from the removedtissue of interest.
 19. The method of claim 11, further comprisingstaging the cancerous tissue.
 20. The method of claim 10, furthercomprising visualizing Cy5 or Cy7 of the selective delivery molecule byan emission ratio imaging method.
 21. The method of claim 10, whereinthe tissue of interest is visualized intraoperatively.
 22. The method ofclaim 10, wherein the molecule is administered intravenously.
 23. Atissue sample comprising a selective delivery molecule of claim
 1. 24.The tissue sample of claim 23, wherein the tissue sample is cancerous.25. The tissue sample of claim 24, wherein the cancerous tissue is:breast cancer tissue, colorectal cancer tissue, squamous cell carcinomatissue, skin cancer tissue, prostate cancer tissue, melanoma tissue,thyroid cancer tissue, ovarian cancer tissue, cancerous lymph nodetissue, cervical cancer tissue, lung cancer tissue, pancreatic cancertissue, head and neck cancer tissue, or esophageal cancer tissue. 26.The tissue sample of claim 24, wherein the cancerous tissue is breastcancer tissue.
 27. The method of claim 24, wherein the cancerous tissueis colorectal cancer tissue.
 28. The tissue sample of claim 24, whereinthe cancerous tissue is squamous cell carcinoma tissue.
 29. The tissuesample of claim 24, wherein the cancerous tissue is skin cancer tissue.30. The tissue sample of claim 24, wherein the cancerous tissue iscancerous lymph node tissue.